Generation of Porcine Angiogenin 4-Expressing Pichia pastoris and Its Protection against Intestinal Inflammatory Injury.

Antimicrobial peptides have been extensively studied as potential alternatives to antibiotics. Porcine angiogenin 4 (pANG4) is a novel antimicrobial peptide in the angiogenin (ANG) family, which may have a regulatory effect on intestinal microflora. The object of present study is obtained pANG4 protein by heterologous expression, so as to explore the biological function of recombinant pANG4 (rpANG4). The pANG4 was expressed in Pichia pastoris (P. pastoris) and anti-inflammatory effects were investigated in intestinal porcine epithelial cell line-J2 (IPEC-J2) and mice. Purified rpANG4 had bacteriostatic activity and did not cause hemolysis or cytotoxicity at concentrations below 128 µg/mL. Purified rpANG4 increased the activity of IPEC-J2 and reduced apoptosis in vitro. rpANG4 reduced the pro-inflammatory gene expression and upregulated tight junction protein gene expression during inflammation. rpANG4 alleviated lipopolysaccharide (LPS)-induced liver and spleen damage, intestinal inflammation, jejunal apoptosis genes' expression, and improved immune function in an in vivo mice model. rpANG4 increased tight junction protein gene expression in jejunum, thereby improving the jejunum intestinal barrier function. In conclusion, rpANG4 had antibacterial activity, inhibited intestinal inflammation, improved intestinal barrier function, and alleviated liver and spleen damage. The current study contributes to the development of antibiotic substitutes and the improvement of animal health.

Establishment of an Indirect ELISA Method for the Detection of the Bovine Rotavirus VP6 Protein.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 µg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

[Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis].

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Expression, purification, and functional verification of recombinant human glycoprotein hormone beta 5/alpha 2 fusion protein in CHO-S cells / 中国药理学通报

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Influence of NDR1 on prognosis of patients with hepatocellular carcinoma and effect on proliferation and migration of hepatocellular carcinoma cells / 安徽医科大学学报

Objective@#To analyze the expression and clinical prognostic significance of nuclear Dbf2-related kinase 1 ( NDR1 ) in hepatocellular carcinoma ，and to investigate the biological function and regulatory mechanism of NDR1 in hepatocellular carcinoma cells．@*Methods@#ENCORI database was used to analyze the correlation of NDR1 and c-Myc．Cycloheximide ( CHX) experiment analyzed the relationship between NDR1 and c-Myc protein stability．The expression levels of NDR1 in liver cancer tissues and normal tissues and its relationship with the survival rate of liver cancer patients were analyzed using the ENCORI database．MYC-NDR1 eukaryotic expression vector was constructed，transfected with hepatocellular carcinoma HepG2 cells，and its expression was verified by protein immuno blotting (Western blot) ; cell proliferation and migration ability were detected by CCK-8 assay，cell clone formation assay and scratch assay，respectively．The correlation between NDR1 and c-Myc expression was analyzed using the ENCORI database，and the relationship between NDR1 and c-Myc protein was investigated using a protein synthesis inhibitor CHX dosing assay. @*Results@#The results of the ENCORI database showed that the expression of NDR1 in liver cancer tissues was higher than that in normal tissues and the overall survival rate of patients with high NDR1 expression was lower than that of patients with low NDR1 expression，and the difference was statistically significant (P＜0. 001) ．The results of the CCK-8 assay showed that the MYC-NDR1 group grew faster than the empty vector group (P＜0. 001) ．The clone formation assay showed that the number of clones in the MYC-NDR1 group was higher than that in the empty vector group (P＜0. 001) ．The cell scratch assay showed that the mean migration distance in the MYC-NDR1 group was greater than that in the empty vector group (P＜0. 001) ．ENCORI database analysis showed that NDR1 correlated with c-Myc expression (R = 0. 184，P＜0. 001) ; CHX dosing assay showed that the reduction of c-Myc protein in the MYC-NDR1 group was lower than that in the empty vector group during the same time．@*Conclusion@#NDR1 is highly expressed in hepatocellular carcinoma tissues，closely correlated with poor prognosis of hepatocellular carcinoma patients ，and positively correlated with the expression of c-Myc gene. The study successfully constructes MYC-NDR1 eukaryotic expression vector ，and the expression product MYC- NDR1 can increase the stability of c-Myc protein and promote the proliferation and migration of human hepatocellu- lar carcinoma cells.

Construction of eukaryotic expression plasmid of human promyelocytic leukemia gene of six transcripts and subcellular location analysis of its recombinant proteins / 中国生物制品学杂志

@#Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.

Eukaryotic expression，purification and activity identification of human His-GRK2 recombinant protein / 安徽医科大学学报

Objective@#To construct a human G protein-coupled receptor kinase 2 ( GRK2) eukaryotic expression system．@*Methods@#The primers were designed ，and the His-GRK2 target gene was amplified by PCR using the Pans-EGFP-GrK2 (full-length) gene as the template．The His-GRK2 target gene was connected to the pcDNA3．1EGFP eukaryotic expression vector． The pcDNA3. 1-EGFP-His-GRK2 plasmid was transfected into HEK 293T cells．48 h later，the expression of GRK2 protein was detected by Western blot，and the GRK2 protein was purified by nickel chelated magnetic bead method．The purification of GRK2 protein was detected by Coomassie bright blue staining and Western blot，and the activity of GRK2 protein was detected by His pull down. @*Results @#The results of double enzyme digestion and sequencing showed that pcDNA3. 1-EGFP-His-GRK2 eukaryotic expression plasmid was successfully constructed．Western blot analysis showed that the molecular weight of GRK2 protein was about 80 ku，indicating that GRK2 protein was successfully expressed in HEK 293T cells (t = 6. 433，P = 0. 003) ．GRK2 protein was purified by nickel chelated magnetic beads．His pull down experiment results showed that GRK2 was bound to prostaglandin E2 receptor 4 (EP4) ，suggesting that GRK2 protein had biological activity (t = 13. 5，P = 0. 000 2) ．@*Conclusion@#The pcDNA3．1-EGFP-His-GRK2 eukaryotic expression plasmid was correctly sequenced and the GRK2 recombinant plasmid was successfully constructed．The GRK2 recombinant plasmid was successfully expressed in eukaryotic cells HEK 293T and the protein expressed was biologically active.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis / 生物工程学报

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Construction and expression of the eukaryotic expression vector of wild-type p27 and lacking the nuclear localization signal p27 / 西安交通大学学报(医学版)

【Objective】 To construct the eukaryotic expression vector carrying the human wild-type p27 and lacking nuclear localization signal p27△NLS coding sequences， and the express them in HEK293T cells， which may contribute to investigating the different locations and roles of p27 in the cytoplasm and nucleus. 【Methods】 Total RNA was prepared from human breast cancer MCF7 cells， and cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR）. After amplification of the p27 CDs and non-NLS fragments by PCR， full length p27WT （CDKN1B， NM_004064.5） and p27△NLS coding regions were obtained. PCR products were then subcloned into the eukaryotic expression vector pCMV-Blank. After identification with bacterial PCR， double restriction enzyme digestion and sequencing， they were defined officially as pCMV-p27WT and pCMV-p27△NLS， respectively. The recombinant plasmids were transfected into HEK293T cells by electroporation. After 48 h， the levels of p27 protein in the cytoplasm and nucleus were detected by Western blotting. 【Results】 The sequencing results showed that the sequences of p27WT and p27△NLS inserted into the plasmids were both correctly consistent with that of NM_004064.5. After transfection with pCMV-p27WT， total p27 protein expression was increased and distributed in both the cytoplasm and nucleus of HEK293T cells. After transfection with pCMV-p27△NLS， p27 protein was significantly increased and almost entirely localized in the cytoplasm of HEK293T cells. 【Conclusion】 The eukaryotic expression plasmids of human p27WT and p27△NLS coding sequences were successfully constructed and overexpressed in HEK293T cells. This research may lay a foundation for investigating the biological function of p27 in the cell cycle progression of tumor cells.

Therapeutic effects of IL-6 and BAFF fusion protein eukaryotic expression plasmid on animal model of Sj?gren′s syndrome / 中华微生物学和免疫学杂志

Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.

Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus / 中国血吸虫病防治杂志

OBJECTIVE@#To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope.@*METHODS@#The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.@*RESULTS@#The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein.@*CONCLUSIONS@#The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Construction of eukaryotic expression vector and bioinformatics analysis of human kidney and brain protein (KIBRA) / 西安交通大学学报(医学版)

【Objective】 To clone the full-length of human kidney and brain protein (KIBRA) coding sequence in eukaryotic expression vector and provide a model for studying the biological function of KIBRA in breast cancer cells. 【Methods】 Total RNA of human breast cancer cell line MCF7 was extracted. After reverse transcription, the full length of KIBRA (NM_001161661.2) coding region was amplified by PCR, and cloned into eukaryotic expression vector pCMV-Blank. After identification, it was defined officially as pCMV-KIBRA. Then it was transfected into MCF7 cells, and the expression of KIBRA was detected by real-time PCR and Western blotting after 48 hours. The primary, secondary and tertiary structures and post-transcriptional modification sites of KIBRA were analyzed with bioinformatics software. 【Results】 Bacterial PCR, double enzyme digestion and DNA sequencing results showed that the correct sequence of KIBRA was inserted into the vector pCMV-KIBRA. The mRNA and protein expressions of KIBRA were significantly increased in MCF7 cells transfected with pCMV-KIBRA. Bioinformatics analysis showed that KIBRA was composed of 1119 amino acids. There were 52 phosphorylation sites, 1 acetylation site and 5 ubiquitination sites, and the protein structure was mainly α-helix and random coil. 【Conclusion】 The eukaryotic expression vector of full-length of human KIBRA coding sequence was successfully constructed and overexpressed in breast cancer cell line MCF7, which can lay a foundation for studying the biological function of KIBRA in breast cancer.

Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox / 中国中药杂志

Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.

Expression and localization of Copine-3-enhanced green fluorescent protein fusion protein / 解剖学报

Objective To construct the eukaryotic expression vector plasmid enhanced green fluorescent protein (pEGFP)-N1-CPNE3, and identify the expression and localization of Copine-3 protein in cells. Methods The Copine-3 coding sequences (CPNE3) was amplified by RT-PCR from human bronchial epithelial (HBE) cells and inserted into eukaryotic expression vector pEGFP-Nl. The recombinant plasmid pEGFP-Nl-CPNE3 was confirmed by endonuclease digestion and sequencing before it was transfected into 293T and H1299 cells. Cellular localization of Copine-3-EGFP fusion protein was detected by con-focal laser scanning. Expression of Copine-3 in 293T and H1299 cells was detected by Western blotting analysis. Localization of Copine-3 in clinical samples of the lung adenocarcinoma patients was detected by immunohistochemistry. Results CPNE3 was successfully constructed into the eukaryotic expression vector pEGFP-Nl and expressed in 293T and H1299 cells. Furthermore, the location of Copine-3 protein in cytoplasm and nucleus was determined by immunofluorescence staining, immuno Western blotting and immunohistochemistry in those cells and clinical samples. Conclusion The eukaryotic expression vector pEGFP-Nl-CPNE3 is constructed successfully, and Copine-3 protein is localized in cytoplasm and nucleus.

Gene cloning, protein expression and examination of biological activity of chicken CD40L / 生物工程学报

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.

DNA Damage-Inducible Transcript 4 (Ddit4) Overexpression Could Inhibit Proliferation and Promote Apoptosis of HT-22 Cells through the Wnt / β-catenin Signaling Pathway / 中国生物化学与分子生物学报

Neural tube defects (NTDs) are congenital defect diseases caused by cell proliferation and apoptosis disorders. Using RNA-Seq assays, we found the increased expression of DNA damage-inducible transcript 4 (Ddit4) in embryonic brain tissues from NTD fetuses. In this study, we intend to explore the effects of Ddit4 overexpression on the proliferation and apoptosis of HT-22 cells and related mechanisms to lay the foundation for the study of the role of Ddit4 in NTDs. According to the mouse Ddit4 sequence, we constructed the eukaryotic expression vector pEX-3-Ddit4. The results of restriction enzyme analysis and sequencing showed that the eukaryotic expression vector pEX-3-Ddit4 was successfully constructed. qRT-PCR and Western blotting results showed that the expression level of Ddit4 in HT-22 cells was significantly increased after transfection of PEX-3-Ddit4 (P < 0. 01) . CCK-8 and Western blotting results showed that Ddit4 overexpression decreased the proliferation of HT-22 cells (P < 0. 01) . Flow cytometry showed that Ddit4 overexpression increased the proportion of cells in the G

Eukaryotic expression, protein purification and biological effects research of human CS1-Fc fusion protein / 生物工程学报

Signaling lymphocyte activation family 7 (SLAMF7/CS1) is a cell surface glycoprotein that is highly expressed in multiple myeloma cells. CS1 is a sensitive and specific biomarker for multiple myeloma. CAR-T cell immunotherapy is a new method for the treatment of multiple myeloma. CS1 CAR-T cell immunotherapy has good effect on relapsed refractory multiple myeloma. To detect the expression efficiency of CS1 CAR on CS1 CAR-T cells and to find an auxiliary means to CAR-T cell immunotherapy, we prepared a CS1-Fc fusion protein. First, the extracellular domain of CS1 was amplified from the existing plasmid by PCR and ligated with human IgG1-Fc fragment by overlap extension PCR. The recombinant fragment was ligated into pMH3 eukaryotic expression vector. After restriction enzyme digestion and DNA sequencing, the pMH3-CS1-Fc-his recombinant plasmid was successfully constructed. The recombinant plasmid was transfected into Chinese hamster ovary cell (CHO-S) by liposome. The expression of the CS1-Fc fusion protein in CHO-S cells was identified by flow cytometry after G418 pressure screening. Next, the CS1-Fc fusion protein was purified by nickel column. Western-blot analysis showed that molecular weight of the fusion protein was about 70 kDa was identified by Western blotting. The CS1-Fc fusion protein couldeffectively detect the expression rate of CS1 CAR and promote the activation, proliferation andcytokines secretion of the CS1 CAR-T cells. The results will lay the experimental foundation for the in vitro detection and potentiation of CAR-T cells in multiple myeloma treated with CS1 CAR-T cell.

Development and application of monoclonal antibodies anti-human lipoprotein-associated phospholipase A2 / 生物工程学报

The aim of this study is to prepare monoclonal anti-human Lp-PLA2 antibodies, and establish a rapid and accurate immunochromatographic Lp-PLA2 assay used in community medical institution. The gene sequence of human Lp-PLA2 was obtained from NCBI to construct the expression plasmid. Lp-PLA2 protein expressed in CHO-K1 cells was used to immune BALB/c mice. The monoclonal antibodies were produced in mouse ascites after hybridoma cells screening. Antibodies were evaluated by SDS-PAGE, ELISA and other methods. The Lp-PLA2 test strip was prepared based on sandwich method and evaluated with the portable detection instrument. The affinity of the paired antibodies, PLA1 and PLA5, both reached 1×10⁻⁸. The antibody subclass was IgG1. Both antibodies recognized the Lp-PLA2 protein in the blood specifically. The Lp-PLA2 test strip was prepared based on sandwich method, with linear range of 20-2000 ng/mL. The Lp-PLA2 test strip correlated well with the diaDexus ELISA test kit. In conclusion, the paired antibodies were successfully prepared with high affinity and specificity. The immunochromatographic test of Lp-PLA2 provided a fast and accurate method to detect the concentration of Lp-PLA2 in blood sample for clinical use in the community medical institution and could contribute to the management of cardiovascular diseases.