Development of a novel anti-human aspartyl-(asparaginyl) ß-hydroxylase monoclonal antibody with diagnostic and therapeutic potential.

Human aspartyl-(asparaginyl)-ß-hydroxylase (HAAH) has recently been the subject of several studies, as it was previously observed to be overexpressed in numerous types of carcinoma cells and tissues in patient tumor samples. HAAH has been implicated in tumor invasion and metastasis, indicating that it may be an important target and biomarker for tumor diagnosis and treatment. However, the immunological tools currently available for the study of this protein, including monoclonal antibodies, are limited, as is the present knowledge regarding the role of HAAH in tumor therapy and diagnosis. In the present study, a recombinant C-terminal domain of HAAH was expressed in Pichia pastoris and a novel monoclonal antibody (mAb) targeting HAAH (HAAH-C) was constructed. Immunofluorescence and antibody-dependent cellular cytotoxicity (ADCC) assays were used to demonstrate the specificity and ADCC activity of this antibody. The results demonstrated that this anti-C-terminal HAAH mAB, in combination with an existing anti-N terminal HAAH mAb, exhibited a high response to native HAAH from carcinoma cell culture supernatant, as measured with a double antibody sandwich enzyme-linked immunosorbent assay. This validated novel mAB-HAAH-C may prompt further studies into the underlying mechanisms of HAAH, and the exploration of its potential in tumor diagnosis and therapy.

[Detection and significance of succinate dehydrogenase of sperm mitochondria].

OBJECTIVE: To detect succinate dehydrogenase (SDH) of sperm mitochondria by an improved method, and to evaluate the relationship between SDH and the motility and viability of sperm. METHODS: In 46 fertile and infertile men (aged from 25 to 36), the motility, viability and mitochondrial SDH of sperm were detected by computer-assisted semen analysis system, dead/live sperm molecular fluorescent probe and an improved cytochemical method. Then, the correlation between the motility and viability of sperm and the positive percentage of sperm mitochondrial SDH were analyzed. RESULTS: In the 46 fertile and infertile men, the motility and viability of sperm were (67.33 +/- 7.37)% and (79.78 +/- 7.65)%, and the positive percentage of sperm mitochondrial SDH was (74.74 +/- 8.29)%. The motility and viability of sperm and positive percentage of sperm mitochondrial SDH had significant correlation (r = 0.901, P < 0.01; r = 0.876, P < 0.01; r = 0.917, P < 0.01). CONCLUSIONS: Detection of sperm mitochondrial SDH has significance in evaluation of sperm mitochondrial function and may serve as an assisted marker of sperm viability.

Inhibition of human natural killer cell functional activity by human aspartyl ß-hydroxylase.

Natural killer (NK) cells are a key component of the innate immune system and play pivotal roles as inflammatory regulators and in tumor surveillance. Human aspartyl ß-hydroxylase (HAAH) is a plasma membrane and endoplasmic reticulum protein with hydroxylation activity, which is over-expressed in many malignant neoplasms and can be detected from the sera of tumor patients. HAAH is involved in regulating tumor cell infiltration and metastasis. Escaping from immune surveillance may help tumor cell infiltration and metastasis. However, the effects of HAAH on tumor immune surveillance have not yet been investigated carefully. The present study investigated the potential use of HAAH as an immune regulator of human NK cells. We assessed the effects of recombinant HAAH (r-HAAH) on primary human NK cell morphology, viability, cytotoxicity, apoptosis, receptors expression and cytokine/cytolytic proteins production. Our results demonstrated that r-HAAH negatively affects NK cell activity in a time and dose-dependent manner. It noticeably reduces the viability of the NK cells by increasing apoptosis and necrosis via caspase signaling pathways. Moreover, r-HAAH reduces the NK cell cytotoxicity by inhibiting surface expression of NKG2D, NKp44 and IFN-γ secretion. These findings suggest that one of the ways by which HAAH actively promotes tumor formation and proliferation is by inhibiting NK cell-surveillance activity.

[Synthesis of artificial antigens of amoxicillin and preparation of monoclonal antibody to amoxicillin].

AIM: To synthesize two kinds of amoxicillin artificial antigen and to establish hybridoma cell lines secreting monoclonal antibody against amoxillin. METHODS: Amoxicillin were respectively coupled with ovalbumin(OVA) and bovine serum albumin(BSA) by using N-hydroxysuc-cinimide active ester (NHS) method to prepared the immunogen and detection antigen. Molar ratio of conjugates were detected by ultraviolet spectrum analysis. BALB/c mice was immunized with AMX-OVA. The hybridomas were obtained by fusing mouse myeloma cells Sp2/0 with splenocytes from the mice immunized with AMX-OVA. The aseites with mAb were purified by octanoic acid-saturated ammonium sulphate method. Isotype was determined by Mouse Isotype kit (Bio-RadLabs.). RESULTS: UV spectra shows overlay features between a small molecular compound and carrier protein. Five hybridoma cell strains secreting specific mAb against amoxicillin were obtained and the five Amb belong to IgG1 kappa isotype. The limit of detection of AMX was 0.25 ng/mL and but there is mild degree of cross-reactions with ampicillin with same structure of ß-lactam ring. CONCLUSION: The anti-AMX Amb could be used to develop immunoassay for detection of residues.

Single-chain variable fragment antibody against human aspartyl/asparaginyl beta-hydroxylase expressed in recombinant Escherichia coli.

The human aspartyl beta-hydroxylase (HAAH) is a highly conserved enzyme that hydroxylates epidermal growth factor-like domains in transformation-associated proteins. Previous studies showed that the gene of HAAH was overexpressed in many human malignancies. In the present study, the HAAH-specific single-chain variable fragment (ScFv) antibody was produced in recombinant Escherichia coli. The variable regions of the genes of the heavy chain (VH) and light chain (VL) cloned from the hybridoma cells G3/F11 were connected with a flexible linker using an overlap extension polymerase chain reaction. Nucleotide sequence analysis revealed that the anti-HAAH VH was a member of the VH V gene family and the VL gene belonged to the Vκ gene family VI subgroup. Extensive efforts to express the functional ScFv antibody in E. coli have been made by using two different prokaryotic expression vectors-pHEN1 and pET-16b-to compare the expression level and solubility of the antibody. The recombinant pHEN1/E1-anti-HAAH vector could express soluble ScFv, although the yield was only 7.8% of the total cellular protein. However, the pET-16b/E2-anti-HAAH vector produced the ScFv as inclusion bodies inside the host cytoplasm, although the expression level of the antibody was quite high (28.5% of the total cellular protein). Soluble ScFv antibody produced by pHEN1/E1-anti-HAAH was characterized for its antigen-binding characteristics. Its antigen affinity as antibody was measured by indirect enzyme linked immunosorbent assay analysis and proved to have high binding activity to the antigen HAAH.

The distribution and expression profiles of human Aspartyl/Asparaginyl beta-hydroxylase in tumor cell lines and human tissues.

Human aspartyl beta-hydroxylase (HAAH) is a highly conserved enzyme that hydroxylates epidermal growth factor-like domains in transformation-associated proteins. Previous studies showed that the HAAH gene was overexpressed in many human malignancies. The present study investigates the distribution and expression profiles of HAAH in a variety of tumor cell lines and human tissues. One hundred and four cases of carcinomas were retrieved from the pathology archives of Department of Pathology, First Affiliated Hospital of Medical College of Xi'an Jiaotong University. Paraffin-embedded tissue specimens were immunostained with the HAAH monoclonal antibodies (MAb) generated to naked plasmid DNA containing N-terminal domain of the encoding HAAH gene and recombinant HAAH. Immunoreactivity was detected in 90.4% (94 positive cases) of carcinomas, including 88.5% of strong positive cases. In addition, HAAH mRNA levels were measured in seven tumor cell lines and in normal tissue. The results demonstrated that HAAH was highly expressed in the tumor cell lines in contrast to its low level of expression in normal tissue. The protein expression level of HAAH in the tumor cell lines by Western bolt analysis was comparable to the mRNA expression level. HAAH distribution in seven tumor cell lines was analyzed by immunofluorescence cell staining. The antigen was primarily localized in cytoplasm and plasmalemma. HAAH mAb exhibited high level binding to the four tumor cell lines (breast carcinoma MCF-7, hepatic carcinoma SMMC-7721, cervix cancer Hela and ovary cancer SKOV) and lower degrees of binding were observed with the other three (renal adenocarcinoma ACHN, bladder cancer BIU-87 and laryngeal cancer Hep-2). In contrast, normal mouse embryonic osteoblasts MC3T3 exhibited no staining. In conclusion, HAAH is overexpressed frequently a variety of carcinomas, indicating that overexpression of the enzyme correlates with the development and progression of carcinomas and it could serve as a prognostic biomarker to predict the clinical course of some carcinomas.

[Cloning of the variable region genes from hybridoma against HAAH and then construction and expression of anti-HAAH scFv].

AIM: Construction and expression of anti-HAAH single chain variable fragment (scFv) by cloning of the variable region genes from anti-HAAH hybridoma cells G3/F11. METHODS: Total RNA was extracted from hybridoma cells G3/F11. By RT-PCR, murine V(H) and V(L) genes of mAb were amplified respectively. Then, They were assembled into V(H)-linker-V(L) scFv template by SOE-PCR and anti-HAAH scFv was express in E.coli by constructed pHEN 1-anti-HAAH vector. The expression of anti-HAAH scFv were detected by SDS-PAGE and Western blotting and the binding activity were demonstrated by ELISA. RESULTS: The analysis of DNA sequencing shown that the full-length of constructed scFv gene was 744 bp, encoding 248 amino acids. Moreover, the V(H) and V(L) genes were functional antibody variable region genes, as there were four FRs and three CDRs in both of them. By SDS-PAGE and Western blotting, the expression level of anti-HAAH scFv were detected. The expression level of pHEN 1-anti-HAAH scFv, which was expressed in E.coli HB2151, was 7.8% in total E.coli protein and were existed in soluble protein mainly. By indirect ELISA detection with HAAH protein, the binding activity of soluble anti-HAAH scFv was very well. CONCLUSION: The murine V(H) and V(L) genes of mAb against HAAH have been cloned successfully and anti-HAAH scFv have been constructed and expressed. Besides, the scFv could be further studied about their biological activity and application, due to their high affinity shown in preliminary detection.

[The distribution and expression pro-files of Aspartyl/Asparaginyl beta-hydroxylase (ASPH) in some tumorous cell lines and tissues].

AIM: To investigate the distribution and expression profiles of Aspartyl/Asparaginyl beta-hydroxylase (ASPH) in some tumorous cell lines and some tissues. To evaluate the distribution and expression profile of Aspartyl/Asparaginyl beta-hydroxylase, ASPH in some tumor cell lines and tumor tissues. METHODS: The expression of ASPH in tumor cell lines on transcriptional level transcriptical level was tested by using RT-PCR, and on translational level was tested by cellular immunofluorescence and Western blot. The histologic sections from tumorous and normal tissue were tested immunohistochemistry. RESULTS: The expression of ASPH could be detected at both the transcriptional level and the translational level in the tumorous cell lines and it distributed in the cell membrane and the cytoplasmic domain. CONCLUSION: ASPH can be wildly used as a potential prognostic marker in neoplasms detection.

Monoclonal antibodies against human aspartyl (asparaginyl) beta-hydroxylase developed by DNA immunization.

We newly cloned the gene encoding the human aspartyl (asparaginyl) beta-hydroxylase (HAAH) from the surgical tissue of a patient with hepatocellular carcinoma. This study was designed to generate HAAH-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were co-immunized with naked plasmid DNA containing N-terminal domain of encoding HAAH gene and recombinant HAAH polypeptide. Hybridomas were developed by the electrofusion of the splenocytes from mice immunized with plasmid DNA to Sp2/0 myeloma cells in vitro. Three hybridoma cell lines (designated G3, G9, and F11, respectively) stably secreting HAAH-specific MAbs were obtained. The specificity and sensitivity of MAbs were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Results showed that the three MAbs belong to IgG1 kappa isotype, the titer of MAbs reached was 5 x 10(4) - 1 x 10(5), and the affinity constant (k(aff)) of MAbs ranged between 2.5 x 10(8) - 1.1 x 10(9). MAb G3 was preliminarily applied to detection expression of HAAH for seven tumor tissues, including hepatocellular carcinoma, lung cancer, kidney cancer, cholangiocarcinoma, prostate cancer, breast cancer, and glioblastoma by immunohistochemical stain. Our studies demonstrated that co-immunization of naked DNA containing encoding gene of target antigen and recombinant target protein, and combined with in vitro electrofusion, is an effective and simple method to raise MAbs.

Sphingosine-1-phosphate and its analogue FTY720 diminish acute pulmonary injury in rats with acute necrotizing pancreatitis.

OBJECTIVE: To investigate the effects of sphingosine-1-phosphate (S1P) and its analogue FTY720 on the lung injury induced by acute necrotizing pancreatitis in rats. METHODS: Acute necrotizing pancreatitis was induced by retrogradely injection of 5% sodium taurocholate of biliopancreatic duct in rats. Sphingosine-1-phosphate (100 microg/kg) or FTY720 (1 mg/kg) was administered immediately after the model induction by peritoneal injection. Six hours after the model induction, bronchoalveolar lavage protein concentration, total cell count, polymorphonuclear neutrophil percentage, proinflammatory cytokines (interleukin 1beta, interleukin 6, and tumor necrosis factor alpha), nuclear factor kappaB activation of alveolar macrophages, and lung myeloperoxidase (MPO) activity were examined. Serum amylase and lipase were tested. In addition, histopathological changes of the pancreas and lung were observed. RESULTS: Bronchoalveolar lavage protein concentration, total cell count, PMN percentage, proinflammatory cytokines, nuclear factor kappaB activation, lung capillary leakage, and lung myeloperoxidase were all reduced significantly in both S1P and FTY720 groups. The pulmonary pathological injury in both S1P and FTY720 groups was ameliorated obviously. Nevertheless, the serum amylase, lipase, and the pancreatic pathological damages were not decreased. CONCLUSIONS: Sphingosine-1-phosphate and its analogue FTY720 significantly decreased pulmonary inflammation and injury in a rat model of acute lung injury caused by acute necrotizing pancreatitis and may represent a novel therapeutic strategy for the acute necrotizing pancreatitis-associated lung injury.

[Study on the application of PEI for gene transfer in mouse skin tissue].

A reliable, low-cost, and highly efficient nonviral gene delivery system using lower molecular weight polyethylenimine (LMW-PEI) is provided. LMW-PEI was linked to an expressing plasmid with green fluorescence protein gene (gfp), the transfection activity mediated by PEIs were examined in the CM7721 cell line and the skin tissue of mouse, respectively. The cytotoxicity of PEIs, the localization and continuance time of gfp expressed in the skin tissue of mouse were also studied. Results showed that the transfection rate of gfp mediated by LMW-PEI in the CM7721 cell line was about 55% . However, with the increasing PEI molecular weight, the cytotoxicity of PEI increased, but its transfection activity decreased. The tissue transfection results showed that LMW-PEI induced a significant expression of the gfp in the cells of hair vesicle and sweat gland of mouse skin tissues following transfection of 24 h, and the expression of gfp lasted 7 - 9 d. When the tissue of mouse was treated with retinoic acid and nitrogenous ketone, respectively, gfp was transferred to the granule layer of mouse skin tissue. The LMW-PEI described here is a new, highly efficient vector; it would be a useful nonviral vector for gene delivery technology.

[Study on membrane adsorption-elution method for concentration of enteroviruses from environmental waters].

A simple and efficient method for concentration of enteroviruses from water sample was established based on the membrane adsorption-elution method combined with eluant concentration. By means of real-time RT-PCR, microporous filters with various nominal pores and materials were compared; the membrane elution method was modified; the effect of PEG on virus recovery was studied; the optimal method was determined at last. In view of the virus recovery and cost, cellulose mixed-ester microporous filter with nominal pore size of 0.22 microm was superior to other filters. Magnetism agitation was used for membrane elution and the optimal final mass concentration of PEG was 130 g/L in eluant concentration step. The recovery of virus was studied in every step in various seeding viruses. The method was tested in surface water, secondary effluent and sewage seeded with viruses. The results showed that the modified method was reliable and suitable for separation and concentration of enteroviruses from various water samples.

[Construction of eukaryotic expression vector expressing hepatitis C virus NS5B and EGFP fusion protein and establishment of stable transfected HepG2 cell line].

OBJECTIVE: To construct a eukaryotic expression vector for expressing hepatitis C virus (HCV) recombinant NS5B-EGFP fusion protein and obtain a stable transfected HepG2 cell line. METHODS: The coding region of NS5B gene of HCV was amplified by PCR and was digested by Xho I/Kpn I. This fragment was inserted into pEGFPN3 with T4 ligase and transformed E. coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectin AMINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level NS5B-EGFP fusion protein was obtained. RESULTS: The eukaryotic expression vector named pEGFPN3-ns5b was successfully constructed and the stable transfected HepG2 cell line expressing NS5B-EGFP fusion protein was obtained. CONCLUSION: The stable transfected HepG2 cell line could express NS5B-EGFP fusion protein, could be used for anti-HCV infection with ns5b gene as the target.

[Identification of oligopeptides mimicking the virus attachment protein of hantaan virus].

OBJECTIVE: To identify and characterize the epitope associated with the virus attachment protein (VAP) of hantaan virus. METHODS: The monoclonal antibody 3G1 was used as the ligand to biospan from a phage-displayed 12-amino acid peptide library, then the positive phage clones were chosen and sequenced. The amino acid sequences of them were compared with that of hantaan virus G2 in homology. The characteristics of positive phage were studied by IFA and ELISA. A decapeptide combining to cell membrane was observed under laser scanning confocal microscope (LSCM). RESULTS: The conservative motif PX(1-2) HX(0-2) H displaying on positive clones shared homologous amino acid sequence with G2 96YPWHTAKCHY105. CONCLUSION: G2 96YPWHTAKCHY105 might play some roles in virus binding to host cell, and might be a possible key epitope of hantaan virus VAP.

[Preparation of specific monoclonal antibody against nucleocapsid protein of SARS coronavirus].

AIM: To express nucleocapsid(N) protein of SARS coronavirus and produce monoclonal antibody(mAb) to N protein. METHODS: N protein gene was amplified by RT-PCR. After being confirmed by DNA sequencing, the gene was subcloned into prokaryotic expression vector. N protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen in the preparation of the mAb. RESULTS: DNA sequencing confirmed that the amplified fragment was N protein gene. SDS-PAGE analysis showed that the M(r) of the expressed protein was approximately 43 kd. This expressed protein could be further confirmed in Western blot by using serum from a convalescent SARS patient as primary antibody. Western blot analysis proved that three mAbs obtained could react specifically to the recombinant N protein. CONCLUSION: The prepared recombinant N protein and mAbs against N protein lay the foundation for further development of early diagnosis assays for SARS coronavirus infection.