Biomedical subjects

Yu-Ying Kong

Publications and source records attributed to Yu-Ying Kong.

8 recordsLinked to original sources

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

[Antiviral activity of a hammerhead ribozyme against HBV in HepG2.2.15 cells].

The carboxy terminal arginine rich domain of core protein is highly conserved among HBV subtypes, and plays important roles in the packaging of pregenomic RNA and viral replication. Therefore, the 3' highly conserved region of HBV C gene is an attractive target for antiviral therapy mediated by ribozymes. A hammerhead ribozyme RzC, targeting the above site, was designed; meanwhile, as controls, the disabled form of RzC, dRzC, was also designed. In order to investigate its antiviral effects in cultured cells, in vitro-transcribed ribozymeRzC, dRzC and ribozyme expression vectors pCRzC, pCdRzC were delivered, respectively, into HepG2.2.15 cells ( clonal cells derived from HepG2 cells that contain integrated HBV ayw DNA). The preliminary results demonstrated that in vitro-transcribed ribozyme RzC had weak inhibition on HBV replication, possibly due to its quick degradation by RNases in cells, while the endogenously expressed ribozyme RzC showed significant inhibitory effect on HBV replication. In conclusion, the results suggested the possibility of the hammerhead ribozyme RzC to be used for the gene therapy of HBV infection.

Antiviral Agents↗

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

Hammerhead Ribozymes Suppress HBV(adr) in HepG2 Cells.

Three hammerhead ribozymes (RS3, RC2 and RC1) targeting to the HBV genome have been designed. Plasmids were constructed by inserting the genes of naked and tRNA-embedded ribozymes into RNA trimming vector pRG523 and then were transferred to eukaryotic expression vector. By the similar cloning method the shotgun-type plasmids carrying homogeneous RS3 or RtS3 unitconnected in tandem were obtained. After co-transfecting the above plasmids and HBV genome containing plasmid into human hepatoma cell line HepG2 respectively and selection by G418, the HBV-inhibiting activity of different kinds of ribozyme in G418-resistant cells was achieved by measuring the decrease of HBV-RNA, progeny DNA and the antigens expressed. The results showed that all the ribozymes were active with more than 70% inhibition activity against the HBV and that tRNA-embedded ribozymes had higher activity than naked ribozymes. It is worth particular interest that shotgun-type ribozymes with the connected unit in tandem with 8 and 12 units constructed in the plasmid revealed the highest activity, reaching >90% inhibition.

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↗

PubMed1999

Roles of Various Regions of the HBV Core Promoter in the Transcription of Precore and Pregenomic RNA.

To determine the potential functions of different regions in the HBV core promoter, several mutants containing B3 and/or C deletion (p3.6IIdeltaB3, p3.5IIdeltaB3, p3.6IIdeltaC, p3.5IIdeltaB3C) have been constructed based on the two HBV transcription units (p3.6II and p3.5II) which contained ENII/Cp and Cp immediate upstream of the linear HBV genome, respectively. The functional effects of deletions were assessed with respect to antigen production, viral mRNA synthesis, and viral DNA replication. When HepG2 cells were transfected with C deletion mutants, HBeAg became undetectable in the supernatant while the expression of HBcAg in the cell lysate was retained. Consistently, precore RNA disappeared but the synthesis of pregenomic RNA and viral DNA replication could still be observed. On the other hand, deletions of B3 fragment reduced the expression of HBeAg and HBcAg significantly, while maintained the synthesis of precore RNA, pregenomic RNA and viral progeny DNA. The results strongly suggest that both fragment C and fragment D serve as minimal promoter elements for the transcription initiation of 3.5 kb RNAs. Notably, the D fragment which contains a novel nuclear factor binding site close to the start site of pregenomic RNA, is critical for the basal transcription of pregenomic RNA, which has not been reported previously. B3 plays an important role in the efficient transcription of precore and pregenomic RNAs, but is not indispensable for their basal transcription.

Journal Article↗

PubMed1996

Regulation of Hepatitis B Virus Gene Expression by Its Two Enhancers.

A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units was constructed. Based on it, a series of mutants with enhancer deletions or point mutations were generated. This system has served as a good model to study the regulation of hepatitis B virus gene expression by its two enhancers (ENI and ENII). The results showed that both ENI and ENII could increase the expression of HBsAg, and the two enhancers worked synergistically. They also could increase the expression of HBeAg, but the deletion of 2C and Ep of ENI had no effect on the expression of Hbe/cAg in the cytoplasm. The expression of C gene might be regulated mainly by ENII, but ENI could cooperate with the action of ENII. The data indicated that ENI and ENII might function differentially but cooperatively in regulation the transcription of the two 3.5 kb mRNAs which functioned distinctively.

Journal Article↗