Structure and Properties of Exopolysaccharide Produced by Gluconobacter frateurii and Its Potential Applications.

An exopolysaccharide (EPS)-producing bacterium was isolated from apricot fermentation broth and identified as Gluconobacter frateurii HDC-08 (accession number: OK036475.1). HDC-08 EPS is a linear homopolysaccharide mainly composed of glucose linked by α-(1,6) glucoside bonds. It contains C, H, N and S elements, with a molecular weight of 4.774 × 106 Da. Microscopically, it has a smooth, glossy and compact sheet structure. It is an amorphous noncrystalline substance with irregular coils. Moreover, the EPS showed surface hydrophobicity and high thermal stability with a degradation temperature of 250.76 °C. In addition, it had strong antioxidant properties against DPPH radicals, ABPS radicals, hydroxyl radicals and H2O2. The EPS exhibited high metal-chelating activity and strong emulsifying ability for soybean oil, petroleum ether and diesel oil. The milk solidification test indicated that the EPS had good potential in fermented dairy products. In general, all the results demonstrate that HDC-08 EPS has promise for commercial applications as a food additive and antioxidant.

Novel Ethyl-3-Aryl-2-Nitroacrylate Derivatives as Potential T3SS Inhibitors against Xanthomonas oryzae pv. oryzae on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a highly destructive bacterial disease. Traditional prevention methods have utilized antibiotics to target bacterial growth, which has accelerated the emergence of resistant strains. New prevention techniques are developing agents such as type III secretion system (T3SS) inhibitors that target bacterial virulence factors without affecting bacterial growth. To explore novel T3SS inhibitors, a series of ethyl-3-aryl-2-nitroacrylate derivatives were designed and synthesized. Preliminary screening of T3SS inhibitors was based on the inhibition of the hpa1 gene promoter and showed no effect on bacterial growth. Compounds B9 and B10, obtained in the primary screening, significantly inhibited the hypersensitive response (HR) in tobacco and the expression of T3SS genes in the hrp cluster including key regulatory genes. In vivo bioassays showed that T3SS inhibitors obviously inhibited BLB and appeared to be more effective when combined with quorum quenching bacteria F20.

Aerobic composting of chicken manure with penicillin G: Community classification and quorum sensing mediating its contribution to humification.

Chicken manure containing antibiotics is a hazardous biological waste. The purpose of our study was to investigate how different concentrations of penicillin G alter the bacterial community to affect humification during aerobic composting of chicken manure. The effect of quorum sensing on the bacterial community was also evaluated. Penicillin G mainly affects low fold changes (within 4) for low-abundance (within 200) microbial genera. We found that the bacterial community cooperated to regulate humus and humic acid synthesis. The microbial genera that make up the bacterial community are different, but each bacterial community may have the same ecological function. Quorum sensing affects humic acid synthesis by regulating carbohydrate metabolism and amino acid metabolism in bacterial communities through mechanisms such as the pentose phosphate pathway and the shikimate pathway. This work presents an understanding of the impact of quorum sensing on the collaboration between bacterial communities during composting.

Diversity-oriented synthesis of fluoroalkylated amines via the palladium-catalyzed divergent fluoroalkylamination of 1,3-dienes.

Herein, we reported the first versatile and expeditious protocol for the diversity-oriented synthesis (DOS) of fluoroalkylated amines via the photoinduced palladium-catalyzed cross coupling of 1,3-dienes, amines and fluoroalkyl iodides, which features excellent 3,4- and 1,4-selectivity controlled by fluoroalkyl iodides, a broad substrate scope as well as good function group tolerance, and could be extended to the late-stage modification of bioactive molecules.

Bacteriocin production and inhibition of Bacillus subtilis by Lactobacillus paracasei HD1.7 in an indirect coculture system.

A broad-spectrum antimicrobial peptide named Paracin 1.7 was produced by Lactobacillus paracasei HD1.7, which was isolated from Chinese sauerkraut juice. In this study, the influence of cocultivation on the communication mechanism of L. paracasei HD1.7 and Bacillus subtilis was investigated. The two bacterial strains were grown in monoculture and indirect coculture, and the growth of both bacteria and bacteriocin production as well as the transcriptional level of luxS in L. paracasei HD1.7 and spo0A in B. subtilis were monitored. Bacteriocin production and cell numbers were increased significantly when L. paracasei HD1.7 cells were indirectly cocultured with B. subtilis, and bacteriocin-producing L. paracasei HD1.7 can prevent the growth and sporulation of B. subtilis. After indirect coculture with B. subtilis, the expression of luxS in L. paracasei HD1.7 increased in the exponential growth phase and decreased in the stationary phase compared to monoculture. The expression of spo0A in B. subtilis dropped in the indirect coculture compared to the monoculture. It indicate that the upregulation of luxS is due to a response to a secreted compound produced by B. subtilis. The results show L. paracasei HD1.7 has an amensalism on B. subtilis, while B. subtilis has a commensalism on L. paracasei HD1.7.

Multiregion sequencing and subclonal analysis reveal intratumoral heterogeneity in esophageal squamous cell carcinoma.

PURPOSE: The aim of this study was to investigate intratumoral genomic heterogeneity and subclonal structure of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: Multiregion whole-exome sequencing was performed on 24 surgically acquired tumor samples from five untreated ESCC patients collected in 2019 to determine the heterogeneity of mutational landscape within tumors. Phylogenetic analysis and mutation process analysis were used to explore the distribution and dynamic changes of mutation spectrum, and subclone analysis was used to explore the subclonal composition and spatial structure of ESCC. RESULTS: An average of 60.2% of mutations were found heterogenous. TP53 and NOTCH1 mutations were confirmed to be early events, and mutations unique in different tumor regions showed a pattern of branching evolution. A large proportion of mutations were associated with abnormal activity of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family, and significant differences in mutation types between trunk and branch variants were found. Subclonal structure exhibited spatial correspondence and spatial limitations, and different genomic features were characterized between close and distant clones. CONCLUSIONS: There is significant intratumoral genomic heterogeneity in the five ESCCs, and their subclonal structure is related to spatial locations.

Radiotranscriptomics signature-based predictive nomograms for radiotherapy response in patients with nonsmall cell lung cancer: Combination and association of CT features and serum miRNAs levels.

PURPOSE: We aimed to establish radiotranscriptomics signatures based on serum miRNA levels and computed tomography (CT) texture features and develop nomogram models for predicting radiotherapy response in patients with nonsmall cell lung cancer (NSCLC). METHODS: We first used established radioresistant NSCLC cell lines for miRNA selection. At the same time, patients (103 for training set and 71 for validation set) with NSCLC were enrolled. Their pretreatment contrast-enhanced CT texture features were extracted and their serum miRNA levels were obtained. Then, radiotranscriptomics feature selection was implemented with the least absolute shrinkage and selection operator (LASSO), and signatures were generated by logistic or Cox regression for objective response rate (ORR), overall survival (OS), and progression-free survival (PFS). Afterward, radiotranscriptomics signature-based nomograms were constructed and assessed for clinical use. RESULTS: Four miRNAs and 22 reproducible contrast-enhanced CT features were used for radiotranscriptomics feature selection and we generated ORR-, OS-, and PFS- related radiotranscriptomics signatures. In patients with NSCLC who received radiotherapy, the radiotranscriptomics signatures were independently associated with ORR, OS, and PFS in both the training (OR: 2.94, P < .001; HR: 2.90, P < .001; HR: 3.58, P = .001) and validation set (OR: 2.94, P = .026; HR: 2.14, P = .004; HR: 2.64, P = .016). We also obtained a satisfactory nomogram for ORR. The C-index values for the ORR nomogram were 0.86 [95% confidence interval (CI), 0.75 to 0.92] in the training set and 0.81 (95% CI, 0.69 to 0.89) in the validation set. The calibration-in-the-large and calibration slope performed well. Decision curve analysis indicated a satisfactory net benefit. CONCLUSIONS: The radiotranscriptomics signature could be an independent biomarker for evaluating radiotherapeutic responses in patients with NSCLC. The radiotranscriptomics signature-based nomogram could be used to predict patients' ORR, which would represent progress in individualized medicine.

[Efficacy enhancement of a Baculovirus-vectored Newcastle Disease Virus F protein vaccine by chicken GM-CSF and IL-2].

To compare with the effects of the GM-CSF and IL-2 used as adjuvants in the baculovirus vaccine, we used genetic engineering to construct the recombinant baculovirus rBV-LMI-F and with GM-CSF and IL-2 to immunized chickens. Then, we compared the concentration of the neutralizing antibody and cytokines to determine the immunostimulatory effects of GM-CSF and IL-2. GM-CSF induced higher levels of antibodies and cytokines in chickens at 28 d and 42 d post-vaccination. In conclusion, GM-CSF could elicit higher serum antibody and cytokines responses and improved the effects of Baculovirus vaccine.

Decreased Siglec-9 Expression on Natural Killer Cell Subset Associated With Persistent HBV Replication.

Siglec-9 is an MHC-independent inhibitory receptor selectively expressed on CD56dim NK cells. Its role in infection diseases has not been investigated yet. Here, we studied the potential regulatory roles of NK Siglec-9 in the pathogenesis of chronic hepatitis B (CHB) infection. Flow cytometry evaluated the expression of Siglec-9 and other receptors on peripheral NK cells. Immunofluorescence staining was used to detect Siglec-9 ligands on liver biopsy tissues and cultured hepatocyte cell lines. Siglec-9 blocking assay was carried out and cytokine synthesis and CD107a degranulation was detected by flow cytometry. Compared to healthy donors, CHB patients had decreased Siglec-9+ NK cells, which reversely correlated with serum hepatitis B e antigen and HBV DNA titer. Siglec-9 expression on NK cells from patients achieving sustained virological response recovered to the level of normal donors. Neutralization of Siglec-9 restored cytokine synthesis and degranulation of NK cells from CHB patients. Immunofluorescence staining showed increased expression of Siglec-9 ligands in liver biopsy tissues from CHB patients and in hepatocyte cell lines infected with HBV or stimulated with inflammatory cytokines (IL-6 or TGF-ß). These findings identify Siglec-9 as a negative regulator for NK cells contributing to HBV persistence and the intervention of Siglec-9 signaling might be of potentially translational significance.

Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity.

Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses.

Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine.

Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens.

In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases.

Effect of small interfering RNA against Paracin 1.7 bacteriocin produced by Lactobacillus paracasei HD1-7.

Lactobacillus paracasei HD1-7 (CCTCCM 205015), isolated from Chinese sauerkraut fermentation broth, contains the bacteriocin Paracin 1.7 which possesses broad-spectrum antibacterial activity. The gene-silencing effect of small interfering RNA (siRNA) is a potential strategy for further understanding the mechanism of production of Paracin 1.7 by L. paracasei HD1-7. In this study, the effect of siRNA on the expression of the most important proteins in the production of Paracin 1.7, sensor kinase (prcK) and response regulator (prcR), was investigated. SiRNA were designed against prcK and prcR, and qRT-PCR was performed to examine the expression of prcK and prcR mRNA. The efficacy of siRNA was determined by comparing the level of antimicrobial activity of the strains. qRT-PCR showed that siRNA-K4 and siRNA-K5 significantly inhibited the expression of prcK mRNA, and siRNA-R4 and siRNA-R6 significantly inhibited the expression of prcR mRNA. The proteins levels and antibacterial activities of mutant strains were lower than the original and control groups, respectively. The results demonstrate that siRNA inhibited both mRNA expression and the production of Paracin 1.7 in L. paracasei HD1-7. Targeting of prcK and prcR with siRNA appears to be a novel strategy for researching the mechanism of Paracin 1.7 production by L. paracasei HD1-7.

Down-regulation of suppressor of cytokine signaling 3 by miR-122 enhances interferon-mediated suppression of hepatitis B virus.

MicroRNA-122 (miR-122) is involved in the pathogenesis of several liver diseases, including chronic hepatitis B infection and hepatocellular carcinoma. This study aimed to explore the potential role of miR-122 in the interferon (IFN)-mediated suppression of hepatitis B virus (HBV) in hepatocytes. We found that elevated expression of suppressor of cytokine signaling 3 (SOCS3) following HBV infection, contributed to the inactivation of the IFN signaling pathway. Based on previous studies from our laboratory showing that miR-122 can modulate type I IFN expression by inhibiting SOCS1 expression, we analyzed the SOCS3 mRNA sequence for putative miR-122 binding sites. We demonstrate that miR-122 inhibits SOCS3 expression by targeting the 3'-untranslated region of the SOCS3 mRNA within the region 1887-1910 nucleotides. Finally, we demonstrate that significantly increased levels of IFN lead to decreased HBV expression in miR-122 mimic-treated Huh7 cells, whereas inhibition of endogenous miR-122 leads to enhanced viral production, owing to a marked decrease in IFN expression. Taken together, our results demonstrate that miR-122 down-regulates SOCS3, thus positively affecting the anti-HBV efficiency of endogenous type I IFN. Our study suggests that suppression of miR-122 induced by HBV infection, leads to the inactivation of IFN expression, which in turn enhances HBV replication, contributing to viral persistence and hepatocarcinogenesis.

[Construction of recombinant baculovirus vectors started by EF1alpha].

OBJECTIVE: To improve the transduction efficiency of baculovirus and exogenous gene expression level, we chose a mammalian cell-specific promoter-human extension factor 1alpha promoter (EF1-alpha), used virus pseudotyped tools--truncated vessicular stomatitis virus protein G (VSV-GED), added woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and adenovirus inverted terminal repeats (ITRs). METHOD: We constructed two improved recombinant baculoviruses transfer vectors named pWK and pWK-ITR with the pFB-VSV-GED-WPRE. The recombinant transfer vectors pWK-eGFP, pWK-ITR-eGFP and pWK (-)-eGFP were constructed by inserting the Enhanced Green Fluorescent Protein (eGFP) reporter gene into the downstream of EF1alpha promoter. Constructed recombinant plasmid transfected Sf9 insect cells, and observed the expression of green fluorescent protein by using the inverted fluorescence microscope. RESULTS: The fluorescence expression rate of BV-WK-eGFP, BV-WK-ITR-eGFP containing WPRE and ITRs was significantly higher than the negative control, ITRs can effectively extend the expression time of eGFP, the expression time of eGFP in BV-WK-eGFP and BV-WK-ITR-eGFP increased 72 hours compared to the negative control BV-WK (-) -eGFP. The transduction time of VSV-GED pseudotyped baculovirus BV-WK-eGFP, BV-WK-ITR-eGFP was obviously shorten in OL cells, and reduced 24 hours compared to the negative control BV-WK (-) -eGFP, transduction efficiency were higher 25.7% and 36.5% than the negative control BV-WK (-) -eGFP, respectively. CONCLUSION: The experiments proved that the VSV-GED could effectively improve the transduction efficiency of baculovirus, WPRE could enhance the expression efficiency of the exogenous gene significantly, and ITRs extend the expression time. The research will lay a foundation to explore improved recombinant baculovirus express exogenous genes in vertebrate cells and research the new recombinant live vector vaccine.

[Construction of baculovirus vector with WPRE regulatory element to express Newcastle disease virus F gene in primary chicken embryo cells].

OBJECTIVE: To construct the recombinant baculovirus with mammaliancell-specific promoter and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), to highly express Newcastle disease virus (NDV) F gene in the primary chicken embryo cells. METHOD: We extracted total RNAs from NDV La Sota strain. Then the F gene was amplified by reverse transcription polymerase chain reaction. We constructed the baculoviral vector (pCMV-WPRE-F) with F gene fused with the WPRE near its 3'end, which expressed under the control of the CMV promoter. The F gene recombinant bacmid was obtained by Bac-to-Bac system and transfected into sf9 insect cells to acquire F gene recombinant baculovirus. After amplification of recombinant baculovirus, the recombinant virus was transfected into chicken primary cells with 50 multiplicity of infection, and the proteins were harvested at 72 h after infection. The F protein expression levels mediated by WPRE regulatory element were analyzed. RESULTS: Western blot results show that the F gene was successfully expressed in chicken primary cells. The product was a 56kDa protein and could be recognized by anti-NDV serum. The WPRE fusion significantly improved the F gene expression as 10 mmol/L butyrate did, but different to butyrate, the WPRE regulatory element was nontoxic to cells. CONCLUSION: The optimized recombinant baculovirus could efficiently deliver NDV F gene into chicken primary cells and express the F antigen protein. In addition, the WPRE regulatory element could increase the expression levels of exogenous gene mediated by baculovirus in chicken primary cells. The research provides us a potential basis for the gene engineered vaccines of NDV and other avian infectious disease based on baculovirus vector.

Optimization of eGFP expression using a modified baculovirus expression system.

The baculovirus gene expression system is an efficient and safe protein expression system, since baculoviruses cannot replicate in mammalian cells. In order to improve the transduction efficiency and increase the reporter gene expression levels delivered by baculoviruses, we tested in the baculovirus expression cassette the Woodchuck hepatitis virus response element (WPRE), and AAV-derived inverted terminal repeats (ITRs) and the truncated vesicular stomatitis virus G protein (VSV-GED). The results showed that WPRE and VSV-GED have synergistic effects and could enhance the expression efficiency of enhanced green fluorescence protein (eGFP), and that ITRs effectively extended the duration of eGFP expression. We also demonstrated that the efficiency of eGFP expression varied under the control of the CMV, CBA, EF1-α or WSSV ie1 promoters in different cell lines.

[Construction of baculovirus vector with Cytomegaloviruse promoter to express eGFP in primary chicken embryo cells].

OBJECTIVE: Baculovirus is known as a safe vector candidate due to its non-replication in mammalian cells. The tropism to different cells and transduction efficiency can be improved by introducing cell-specific promoter, VSV-GED and different functional regulatory elements. The optimized pseudotyped recombinant baculovirus can express eGFP gene in primary chicken cells, which provides us a new approach to develop engineered poultry vaccines. METHOD: The pseudotyped recombinant baculoviruses were constructed with cytomegaoviyns (CMV) promotor, VSV-GED, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and inverted terminal repeats (ITRs). The recombinant baculoviruses contained eGFP reporter gene were transfected chicken primary cells, and the eGFP protein expression levels mediated by different baculoviruses were analyzed. RESULTS: The expression of eGFP was detected at 12 hours after infection. The transduction efficiency of the pseudotyped recombinant baculoviruses increased from 36% to 48.2% by inserting VSV-GED. The expression effect of eGFP in recombinant baculovirus carrying WPRE element was similar to that by adding 10 mmol/L butyrate. However, the WPRE elements are nontoxic to cells. Within 72 hours, the expression intensity of eGFP in the recombinant baculovirus with ITRs increased gradually. CONCLUSION: The VSV-GED element can improve the transduction efficiency and WPRE can increase the reporter gene eGFP expression levels mediated by baculovirus in chicken primary cells. The recombinant baculovirus with the ITRs elements can extend the expression time of eGFP.

[Construction of BV-T7 hybrid expression system based on baculovirus to express target gene eGFP in mammalian and chicken cells].

OBJECTIVE: To investigate whether the recombinant baculovirus BV-T7 hybrid expression system can be effectively transduced into chicken cells in vitro, and whether it can express the foreign genes (eGFP). METHOD: We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells and chicken cells. Two recombinant baculoviruses were constructed, one carrying cDNA of bacteriophage T7 RNA polymerase, with a nuclear localization signal, under the control of a mammalian promoter and the other expressing eGFP gene under the control of T7 promoter. The constructed recombinant baculoviruses co-infected mammalian oligodendrocyte cells, as well as chicken embryo fibroblasts cells and chicken embryo skeletal muscle cells. RESULTS: The eGFP activity was detected in mammalian cell lines and embryo fibroblasts cells and chicken embryo skeletal muscle cells. The recombinant baculovirus transduction efficiency of oligodendrocyte cells was 59.5%, and in CEF cells and myoblast cells the transduction efficiencies were 23.2% and 33.1%. CONCLUSION: BV-T7 hybrid expression could be expressed T7 RNAP in mammalian cells and avian cells.

[The expression of IBDV VP2 in chicken primary myoblast cells using the baculovirus vector].

OBJECTIVE: To construct the recombinant baculovirus expressing Infectious bursal disease (IBDV) VP2 gene in the chicken primary myoblast cells. METHODS: A proteinase K digestion and phenol-chloroform extraction method was used to extract dsRNA genome from IBDV. VP2 gene was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) with the genome RNA as template. The pFastBac-pCMV-VP2 baculovirus transfer vector was constructed by inserting VP2 gene under the immediate-early promoter of cytomegalovirus. The VP2 recombinant bacmid was obtained by Bac-to-Bac system and transfected sf9 insect cell to acquire VP2 recombinant baculovirus. After amplification of recombinant baculovirus on cell passages, the recombinant virus was seeded on chicken primary myoblast cells with 50 multiplicity of infection (MOI), and the cells were harvested at 72 hours after infection. RESULTS: Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) and Western blot results showed that the VP2 gene was successfully expressed in chicken primary myoblast cells. The product was a 48kDa protein and could be recognized by anti-IBDV serum. CONCLUSION: The recombinant baculovirus could efficiently delivery IBDV VP2 gene into chicken primary cells and that CMV, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct the expression of VP2 antigen protein. The research can be a potential basis for the development of baculovirus vector vaccines for IBDV and other avian infectious disease.