Biomedical subjects

Li-Xin Zhu

Publications and source records attributed to Li-Xin Zhu.

11 recordsLinked to original sources

PubMed2005

Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli.

AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli (E. coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni(2+)-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 glycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.

Animals↗

PubMed2004

Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells.

AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis. METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO) cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258 staining, flow cytometry and DNA fragmentation analysis. RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line. CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

Animals↗

PubMed2004

A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses.

AIM: To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine. METHODS: Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes. These cells were subjected to HCV antigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals. RESULTS: Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-gamma secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-gamma but did not change the profile of IL-4 secretion. CONCLUSION: Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

Adjuvants, Immunologic↗

PubMed2004

Elasticity of small artery in patient with spontaneous rupture of hepatocellular carcinoma.

Objective: Spontaneous rupture of hepatocellular carcinoma (HCC) is common in Asia and Africa with unclear mechanism. In our previous study, we found that the vascular injury was related to the HCC rupture. In this study, the structure of elastin around the small artery was deeply investigated to confirm our previous study. Methods: Immunohistochemical technique and transmission electron microscopy were used to study 23 specimens from ruptured HCC and 30 cases with nonruptured HCC. Results: The layer of elastin around the vascular wall was significant thicker in patients with ruptured HCC than that in nonruptured HCC. The proliferation of elastin, abnormal distribution of neutrophil elastase and degradation of collagen fibril were predominantly present in the specimens from ruptured HCC. The phenomenon that the infiltrated neutrophils from bloodstream into the vascular wall, which caused the vascular injury, can be found in specimens from ruptured HCC. The vascular injury mainly occurred in small artery. Since the damaged vessels could become stiff and weak, which would be more prone to splitting and result in hemorrhage in patients with ruptured HCC, we postulated that the vascular injury, especially the inelastic small artery, may relate the ruptured HCC. Conclusion: The vascular injury in small artery might relate to ruptured HCC.

Journal Article↗

PubMed2002

Ultrastructural study of the vascular endothelium of patients with spontaneous rupture of hepatocellular carcinoma.

BACKGROUND: Spontaneous rupture of hepatocellular carcinoma (HCC) is common in Asia and Africa, although the mechanism is unclear. In our previous study, we found that vascular injury exemplified by collagenase synthesis and collagen degradation in small arteries was related to the HCC rupture. METHODS: In this study, transmission electron microscopy was used to study 22 specimens from ruptured HCC and non-ruptured HCC. RESULTS: In nine specimens of ruptured HCC, there was evidence of vascular injury with fewer cell junctions and larger fenestrae in vascular endothelial cells. The phenomenon of increased endothelial protein synthesis was also present. In the specimens of non-ruptured HCC, evidence of vascular injury was found in only two cases (p < 0.01). Fewer cell junctions and larger fenestrae could increase the permeability of the vascular wall. Increased protein synthesis in endothelial cells correlates with the phenomenon that more collagenase is expressed in these cells. The resulting breakdown of collagen could render the blood vessels weak; hence, these blood vessels are more prone to splitting/breakage. CONCLUSION: We conclude from our study that this vascular injury may result in HCC rupture.

Adult↗

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

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↗

PubMed2002

Cartilage end-plate rupture of the lumbar vertebrae: report of 7 cases.

The authors report the clinical manifestations, imaging features, surgical approaches and the outcome of 7 cases of cartilage end-plate rupture of the lumber vertebrae, attempting to explore the pathogenesis and describe the clinical and pathological features of the disease. The diagnosis and treatment are also discussed in brief.

Adult↗

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↗

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↗