Biomedical subjects

Guang-Di Li

Publications and source records attributed to Guang-Di Li.

13 recordsLinked to original sources

PubMed2002

Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay.

AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.

Amino Acid Sequence↗

PubMed2002

Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein.

AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively. Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

Animals↗

PubMed2002

DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV.

AIM: Both Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are major causative agents of transfusion-associated and community-acquired hepatitis worldwide. Development of a HCV vaccine as well as more effective HBV vaccines is an urgent task. DNA immunization provides a promising approach to elicit protective humoral and cellular immune responses against viral infection. The aim of this study is to achieve immune responses against both HCV and HBV by DNA immunization with fusion constructs comprising various HCV E2 gene fragments fused to HBsAg gene of HBV. METHODS: C57BL/6 mice were immunized with plasmid DNA expressing five fragments of HCV E2 fused to the gene for HBsAg respectively. After one primary and one boosting immunizations, antibodies against HCV E2 and HBsAg were tested and subtyped in ELISA. Splenic cytokine expression of IFN-gamma and IL-10 was analyzed using an RT-PCR assay. Post-immune mouse antisera also were tested for their ability to capture HCV viruses in the serum of a hepatitis C patient in vitro. RESULTS: After immunization, antibodies against both HBsAg and HCV E2 were detected in mouse sera, with IgG2a being the dominant immunoglobulin sub-class. High-level expression of INF-gamma was detected in cultured splenic cells. Mouse antisera against three of the five fusion constructs were able to capture HCV viruses in an in vitro assay. CONCLUSION: The results indicate that these fusion constructs could efficiently elicit humoral and Th1 dominant cellular immune responses against both HBV S and HCV E2 antigens in DNA-immunized mice. They thus could serve as candidates for a bivalent vaccine against HBV and HCV infection. In addition, the capacity of mouse antisera against three of the five fusion constructs to capture HCV viruses in vitro suggested that neutralizing epitopes may be present in other regions of E2 besides the hypervariable region 1.

Animals↗

PubMed2002

Expression and characterization of hepatitis C Virus E2 glycoprotein fused to hepatitis B virus preS1(21-47) fragment in CHO cells.

To stably express hepatitis C virus (HCV) E2 glycoprotein in CHO cells and facilitate the detection and purification of the expression products, the gene fragment encoding N-terminal 277 amino acids of this protein was fused to the fragment encoding hepatitis B virus (HBV) preS1(21-47) region and inserted into a secretion vector pSecTagB. CHO cells transfected with the recombinant plasmid carrying fusion gene were selected under growth pressure of Zeocin. Secreted fusion products and its cell-associated counterpart were detected by Western blot using E2 specific or preS1 specific antibodies. Glycans carried by the expression products were analyzed with glycan-type specific glycosidases. Most of the cell-associated E2 were found to be high-mannose-type glycosylated, while the secreted E2 proteins were found to be mostly complex-type glycosylated, suggesting further modification in Golgi apparatus upon secretion. Primary studies showed that the fusion antigen could be specifically bind to and elute from anti-preS1 antibody coupled Sepharose resin, suggesting that large-scale preparation of the fusion antigen is feasible with an immunoaffinity resin. This work will contribute to the further study of immunological properties of HCV E2 glycoprotein and also to the study of recombinant HBV/HCV vaccine.

Animals↗

PubMed2002

DNA immunization with recombinant HCV E2 expression plasmids.

DNA-based immunization, the delivery of plasmid DNA by direct inoculation, is a newly developed method of vaccination. Besides of many advantages, DNA immunization was demonstrated to generate weaker antibody and CTL responses than did protein and live attenuated vaccines, respectively. To circumvent this shortage, several methods were tested such as using different vector, changing injection mode, and co-expressing cytokines, etc. The studies showed that many factors could greatly affect the immune responses to DNA vaccine. The aim of this study is to compare different vectors for the presentation of the HCV E2 to generate immune responses by using DNA-based immunization. Four expression plasmids, with different promoter types and with or without signal sequences were constructed, which encode C-terminally truncated E2 (384-660) with HBV preS1 21-47 tag fused to its N-termini. Transient expression in HeLa cells showed that only recombinant plasmids with signal sequence could be expressed and properly processed, and the product could be secreted into medium. Protein expression level is slightly higher in plasmids with CMV promoter than with EF1alpha promoter. After immunization of C57BL/6 mice, all the recombinant constructs could elicit both anti-preS1 and anti-E2 antibodies. But only pCMV Sec-S1E2t660 with both CMV promoter and signal sequence could induce high-level and long-lasting antibody, showing that it is a good vaccine candidate. Possible reasons for the different immune responses to these constructs were discussed.

Animals↗

PubMed2001

High Frequency of Homologous Recombination in the Genome of Modified Vaccinia Virus Ankara Strain (MVA).

MVA is a genetically modified vaccinia virus strain, which is replication defective and extremely safe for research and clinical use. To further improve the safety of MVA and achieve more efficient selection of recombinant constructs, a transient selection marker system has been developed, which contains vaccinia virus k1l gene flanked by two identical fragments. In this report, four recombinant MVAs were constructed with this system, and homologous recombination of recombinant MVAs was studied during the construction procedure. The results showed that the recombination frequency of these rMVAs was significantly high, though lower than that observed in other vaccinia virus strains. The k1l free rMVAs could be conveniently obtained after 3 or 4 blind passages. Our data indicated that recombinant MVA with a transient selection marker system was safe for use as live vaccine and gene therapy vector for human. In addition, blind passages could enhance the efficiency in isolation of k1l free recombinants

Journal Article↗

PubMed2001

Expression, Purification and Preliminary Clinical Use of Recombinant HBsAg GST-PreS1(21--47 aa) Fusion Proteins.

Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21--47 aa) DNA fragment, respectively, were constructed. GST-PreS1(21--47 aa) and GST-2xPreS1(21--47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG. The expression level of GST-PreS1(21--47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST-2xPreS1(21--47 aa) was about 15% of total soluble proteins, asestimated by SDS-PAGE. 50 mg GST-PreS1(21--47 aa) or 20 mg GST-2xPreS1(21--47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione-Sepharose 4B. Direct ELISA results showed that antigenicity of GST-2xPreS1(21--47 aa) was better than GST-PreS1(21--47 aa) and synthetic peptide. Using GST-2xPreS1(21--47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti-PreS1(21--47 aa) antibody, based on protein A-biotin and streptavidin-HRP, was established. The results from 99 sera samples of hepatitis B patients showed that anti-PreS1(21--47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients. Clinical follow-up study suggested that appearance of anti-PreS1(21--47 aa) was related to the course of the disease and recovery of patients. Detection system established in the study is promising for clinical application.

Journal Article↗

PubMed2000

Purification and Characterization of Recombinant Hepatitis B Virus Surface Antigen SS1 Expressed in Pichia pastoris.

The purification of recombinant hepatitis B surface antigen, SS1 protein, expressed in Pichia pastoris, and the investigation of its physiochemical characters and immunogenicity were described here. Employing McAb immunoaffinity chromatography, this protein was purified to purity of 95%. The results of ELISA and Western blotting showed good antigenicity of this purified SS1 protein. CsCl gradient centrifugation and electron microscopy assay proved that this purified protein could be assembled into particles similar to the HBV subviral particles. Strong antibody responses against both the HBs and PreS1 epitopes were induced in BALB/c mice immunized with this purified protein. The simultaneous injection of a CpG adjuvant induced a Th1-like immune response against both the HBs and PreS1 epitopes.

Journal Article↗

PubMed2000

Expression of the Recombinant Hepatitis B Virus Surface Antigen Carrying PreS Epitopes in Pichia pastoris.

Many studies have suggested that hepatitis B surface antigen(HBsAg) including PreS sequences could be an ideal candidate for highly effective hepatitis B virus vaccine. Modified surface antigens S1S, SS1 and S2S which carried PreS epitopes, were expressed in Pichia pastoris. The characterization of antigenicity and particle assembly demonstrated that the expression products could be assembled into particles which presented S, PreS1 or PreS2 antigenicity, respectively. The expression was more efficient than that in Saccharomyces cerevisiae.

Journal Article↗

PubMed1999

Fusion Expression, Immunogenicity and Applications of C-terminally Truncated HCV Core Proteins.

Clones expressing full-length(aa 1-191) or C-terminally truncated forms (aa 1-69 and aa 1-40) of hepatitis C virus core(HCc) protein fused to the C-terminal of glutathione S-transferase(GST) were constructed and their expressions in different strains of Escherichia coli were compared. The expressed proteins were soluble. ELISA and Western blot analyses showed that GSTC191 was poorly expressed and had poor stability, whereas GSTC69 and GSTC40 were stable and could be purified to 90% purity in a single step on glutathione-Sepharose 4B. Mice immunized with these purified proteins produced high-titre antibodies. As an application, purified GSTC69 and GSTC40 were used in a preliminary assay of anti-HCc antibodies in patients'sera, and results showed that they had high specificity in the assay.

Journal Article↗

PubMed1996

The Fusion Expression of HBV preS Epitopes and the Core Antigen.

The DNA fragments encoding the preS epitopes of HBV surface antigen were fused to the HBc gene and expressed in E. coli under the control of the tac promoter. The products were analyzed by ELISA and Western blotting, which confirmed that the hybrid proteins were expressed as expected. Analysis by electron microscopy and CsCl density gradient ultracentrifugation revealed that all fusion proteins were able to form particles, only with a slightly lower density than the native multimeric HBc. Partially purified fusion particles were then used as immunogen to Balb/c mice and high titer antibody against the preS1(21-47) epitope was observed, which demonstrated that the immunogenictiy of preS1 (21-47) could be greatly improved when fused in the el loop in HBc protein.

Journal Article↗

PubMed1996

Affinity Purification of Hepatitis B Virus Surface Antigen Containing PreS1 Region.

The large protein of hepatitis B virus surface antigen (LHBs) contained an attachment site of HBV to liver cells and the antibodies to preS1 were virus-neutralizing. Therefore, vaccines containing preS1 would be more protective. However, One of the key problems in the preparation of gene-expressed proteins was the purification of the products. We have established a method of immuno-affinity chromatography with the anti-HBsAg-preS1 monoclonal antibody. Using this technique, We have successfully purified gene-expressed fusion protein of hepatitis B virus surface antigen containing preS1 (21-47). Results showed that this method was simple, effective and specific as compared to other methods, and might be useful in the future.

Journal Article↗

PubMed1996

The Expression of HCG Epitope Fused to Hepatits B Virus Core Antigen.

The DNA fragments encoding HGC-beta-37-CTP was amplified by PCR and fused to the core gene of HBV at the position of amino Acid 1(N-terminal fusion, pCn-HCG), 154(C-terminal fusion, pCc-HCG), 75-83(internal fusion,pCm-HCG), and both 75-83 and 154 (pC-HCG2) respectively. The fused genes were expressed in E. coli, and the antigenicity of both HBcAg and HCG as well as the expression level were analyzed. In addition, the chimeric particular characteristics of the proteins and their immunogenicity were identified. It revealed that the fusion proteins pCm-HCG and pCc-HCG were able to form particles, and that the fusion protein pCm-HCG could induce antibody of anti-HCG of high titers in mice, suggesting that the position at 75-83 amino acid residue should be a relatively promising fusion site for HCG.

Journal Article↗