Antigenic analysis of two chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.</p><p><b>METHODS</b>PreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.</p><p><b>RESULTS</b>The morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.</p><p><b>CONCLUSION</b>Chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.</p>

Construction and characterization of hepatitis B surface antigen "a" epitope virus-like particles / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface.</p><p><b>METHODS</b>Hepatitis B surface antigen "a" epitope were inserted into the Hepatitis B core antigen, between the 78th (Asp) and the 79th (Pro) amino acids. The gene was synthesized after the codon optimized, then it was ligated to the express vector after been enzyme digest. The virus-like particles were observed by electron microscope and detected by ELISA after been expressed and purified. Immune the rabbits by the VLPs, then detect the antibody.</p><p><b>RESULT</b>The virus-like particles were confirmed by electron microscope. Its antigenicity and immunogenicity were identified by ELISA.</p><p><b>CONCLUSION</b>The prokaryotic express plasmid with the fusion gene has been constructed successfully. The virus-like particles have been expressed, purified and identified, which lays the foundation for its application in the further.</p>

Expression of hepatitis C virus subunit fusion protein and analysis of its immunogenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Obtain the hepatitis C virus high purified subunit fusion protein and detect its immunogenicity.</p><p><b>METHODS</b>With the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21 (DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein. At the same time, the sera were collected and prepared from the immunized experimental animals in order to investigate the immunogenicity of the protein by EIA.</p><p><b>RESULTS</b>High purified hepatitis C virus subunit fusion protein was obtained successfully. The EIA indicated that the fusion protein could elicit specific antibodies in the animals with very high titers.</p><p><b>CONCLUSION</b>The hepatitis C virus subunit fusion protein expressed in prokaryotic system was proved to have strong immunogenicity. It could provide some helpful and useful information to the hepatitis C virus prophylactic and therapeutic vaccine development.</p>

Preparation and identification of the monoclonal antibodies against programmed death-1 ligant 1 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (McAbs) against human programmed death-1 ligant 1(PD-L1) and identify its bioactivity.</p><p><b>METHODS</b>We immunized the BALB/c mice with Thioredoxin-(PD-L1) recombination protein which expressed by prokaryotic system. Prepare hybridoma cell by hybridoma technology and used enzyme-linked immunosorbent assay(ELISA) and Western-blotting assays to select positive hybridoma identify cell. Competition inhibition ELISA was carried out to identify the special bioactivity of antibody.</p><p><b>RESULTS</b>4 hybridoma cell strains which could secrete anti-(PD-L1) antibodies stably were selected. The McAbs has good affinity with its receptor. Purify anti-(PD-L1) with title 1:32 000 was obtained after large quantity preparation. At the same time we obtained 1 cell stain which could secret special anti-Trx McAbs.</p><p><b>CONCLUSIONS</b>We obtained anti-(PD-L1) McAbs with good bioactivity successfully, which lay the foundation for further study.</p>

Preliminary study on genotype of hepatitis B virus detected from Xinjiang Uygur Autonomous Region in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To determine the main genotype of hepatitis B virus (HBV) in Xinjiang.</p><p><b>METHODS</b>200 HBsAg positive serum specimens were detected from more than 2000 serum of Xinjiang inhabitants, and HBV S gene was detected by using nPCR amplifying, and compared with the standard S region HBV nucleotide sequences of genotypes A-H retrieved from GenBank, then analyzed and drawn the polygenetic tree by MEGA3 software.</p><p><b>RESULT</b>Gene in 127 (63.5%) serum specimens was detected from 200 samples. Among 127 serum specimens, 10 (7.8%) was genotype B, 58 (45.7%) was genotype C, and 59 (46.5%) was genotype D.</p><p><b>CONCLUSION</b>Genotype B, C and D have been found in Xinjiang.</p>

Analyze the bioactivity of PD-1 and PD-L1 recombination protein which expressed by prokaryotic system / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the interaction of PD-1 and PD-L1 recombination protein and to know their bioactivity and affinity.</p><p><b>METHODS</b>Stick the PD-1 protein on the surface of CM5 sensor chip by the method of Ammine coupling after being preconsentrated. Dilute the PD-L1 protein step by step and reject it to the passage on CM5 sensor chip which had been stick by PD-1. The time of combination is 3 minutes and of separation is 15 minutes, respectively. Observe the procession and analyze data by BIA Evaluation software 4.</p><p><b>RESULTS</b>On the consistency of 40 microg/ml, pH 4.5, the PD-1 protein could couple steady on the surface of CM5sensor chip, RU is 3300. On the density of 200 mmol/ml PD-L1 could combine with PD-1 specifically, RU = 150, K(D) = 3.5 x 10(-6).</p><p><b>CONCLUSION</b>The PD-1 and PD-L1 recombination protein which we expressed by prokaryotic system have good affinity and bioactivity. The results could provide basic condition for later study.</p>

Express the recombinant protein PD-L1 in prokaryotic and analyze its biological activity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the programmed cell death 1 ligant 1 (PD-L1) recombination expression vector, express the fusion protein in prokaryotic and analyze the biological action of express product.</p><p><b>METHODS</b>The whole PD-L1 gene sequence was synthesized after codon optimized. Construct the thioredoxin-(PD-L1) recombination expression vector and express the fusion protein in E. coli. Purified the target protein and analyze the conjugated ability of protein by ELISA.</p><p><b>RESULTS</b>The PD-L1 recombinant expression vector has been constructed correctly. The target protein has been obtained with which expressed in high efficiency and production. The target protein can conjugate specifically with the PD-1, its specific receptor.</p><p><b>CONCLUSION</b>We have obtained the PD-L1 recombinant protein success with high biological activity. The result provide the basic condition for further study on antibody and mutually action between PD-L1 and chronic virus infectious.</p>

Preparation and identification of the monoclonal antibodies against VP1 capsid protein of Enterovius 71 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies (McAbs) against VP1 capsid protein of Enterovirus 71.</p><p><b>METHODS</b>Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, were synthesized respectively. Immunized BALB/c mice with the synthetic peptides to establish the hybridoma cell strains secreting specific McAb to VP1. After the specific McAbs were prepared, identified and analyzed the titer by indirect ELISA assay. The positive clones were selected and their neutralization titer were determined by neutralization test.</p><p><b>RESULTS</b>Two high titered anti-VP1 antibodies secreted by the hybridoma cells showed good neutralization reaction with enterovirus 71 on RD cells, and the neutralization titer were 1:8 and 1:16 respectively.</p><p><b>CONCLUSION</b>Two high titered anti-VP1 antibodies, with good neutralization activity, secreted by the hybridoma cells, which lays the foundation for further study.</p>

Expression of recombinant rubella virus E1 protein and initial application for detecting of antibody / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To apply recombinant rubella virus envelope protein-1 (E1) to detect human rubella virus IgG antibody.</p><p><b>METHODS</b>Rubella virus E1 protein was expressed in E. coli, purified E1 protein was used as the antigen for the detecting of anti rubella in human sera in the way of enzyme linked Immunosorbant assay (ELISA).</p><p><b>RESULTS</b>The antigenicity of the recombinant protein was checked by WHO rubella sera panel. We detected 200 sera samples, which came from Guangxi Guilin. 93% of these samples were positive.</p><p><b>CONCLUSION</b>The antigenicity of recombinant E1 is a satisfied candidate antigen for the detecting of human rubella virus antibody. The prevalence of anti rubella virus IgG in Guangxi is 93%. It is at the some level compared with other provinces in China.</p>

Protection effects of acetylcholinesterase inhibitors on apoptosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate whether tacrine and donepezil can prevent apoptosis induced by Lipopolysaccharides.</p><p><b>METHODS</b>Phase-contrast microscopes was used to observe the morphological changes of Vero cells. Cell counting kit-8 was used to measure cell survival vitro. DNA fragment was analyzed by agarose gel electrophoresis to determine the apoptotic biochemical changes.</p><p><b>RESULTS</b>Vero cells treated with 400 microg/ml and 500 microg/ml Lipopolysaccharides exhibited cell apoptosis. 10 micromol/L tacrine provided protective effect to 500 microg/ml Lipopolysaccharides induced cell apoptosis measured by Phase-contrast microscopes, cell counting kit-8 and DNA fragment analyze. However, donepezil did not show any protective effect of the apoptosis induced by 500 microg/ml Lipopolysaccharides.</p><p><b>CONCLUSION</b>Lipopolysaccharides can induce apoptosis in Vero cells to built an apoptotic model in vitro. Tacrine rather than donepezil can inhibit Lipopolysaccharides induced apoptosis in Vero cells.</p>

Cloning of PD-1 gene and its prokaryotic expression in Escherichia coli / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli.</p><p><b>METHODS</b>The human PD-1 cDNA was cloned by RT-PCR from the total RNA, which was extracted from peripheral blood lymphocyte cell of the patient with chronic hepatitis B. Recombinant PD-1 protein was been expressed and purified after the prokaryotic expression plasmid had been constructed. It was identified by SDS-PAGE, DNA sequencing and amino acid sequencing.</p><p><b>RESULTS</b>The PD-1 gene was cloned and confirmed by DNA sequencing. The recombinant protein was expressed in E. coli. The purified protein was obtained, then been confirmed by amino acid sequencing.</p><p><b>CONCLUSION</b>The human PD-1 gene was successfully cloned and expressed in E. coli, which lays the foundation for further study on the function and application of PD-1.</p>

Antigenic properties of mutant hepatitis B virus surface antigen / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the antigenic properties of mutant hepatitis B virus surface antigen, to understand the sensitivity of the commercially available HBsAg assays to the variants and to reduce the undetectability of the variants.</p><p><b>METHODS</b>Recombinant eukaryotic expression plasmids for HBsAg. The recombinant eukaryotic expression plasmids pSS1adr, pSS1adw2, pSS1adw2- 145Arg, pSS1adr-126 Asn and pSS1adr-126Ser were transfected into COS-7 cells. HBsAg in the supernatants of transfected cells was detected by using different commercial ELISA kits.</p><p><b>RESULTS</b>The absorbance value of pSS1adr-126 Asn and pSS1adr-126Ser plasmids were similar to that of the wild type HBsAg, the absorbance value of pSS1adw2-145Arg plasmids was lower than that of the wild type HBsAg.</p><p><b>CONCLUSION</b>It is estimated that the antigenicity of HBsAg mainly depended on the amino acid sequence of "a" antigen determinant and its conformation, so 145 amino acid substitutions led to the change of conformation and the antigenicity of variant HBsAg was lower than that of the wild type.</p>

Genotypes of hepatitis B virus in Henan Luohe area / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To determine the main genotype of hepatitis B virus (HBV) prevailed in Henan Luohe area.</p><p><b>METHODS</b>Serum specimens were collected from 94 HBsAg positive individuals, and HBV S gene were obtained by PCR amplifying, and the gene sequences were analyzed and the polygenetic tree was drawn by the software MEGA3.</p><p><b>RESULTS</b>About 75.7% samples of HBV S gene clustered in genotype C, about 20% samples clustered at genotype B in the HBV polygenetic tree, about 4.3% samples clustered at genotype D in the HBV polygenetic tree.</p><p><b>CONCLUSION</b>The main genotype of hepatitis B virus prevalent in Henan Luohe is genotype C, genotype B is rarely seen, and genotype D is rarely seen.</p>

Expression of the P1B, P2A, P3AB and P3D protein of hepatitis A virus in prokaryotic cell and antigenicity analysis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express P1B, P2A, P3AB and P3D cDNA gene fragments in prokaryotic system using thioredoxin fusion expression system; to investigate the antigenicity and application of recombinant protein.</p><p><b>METHODS</b>By using PCR technique, P1B, P2A, P3AB and P3D gene fragments were cloned. Choosing M47 as the expressive vector, the recombinant plasmid P1B, P2A, P3AB and P3D was constructed and expressed in Escherichia coli after inducing by IPTG. By anion exchange and affinity chromatography, purified recombinant protein was obtained. By using Western Blot analysis and indirect ELISA to detect its antigenic activity.</p><p><b>RESULTS</b>Four recombinant plasmids was proved to be constructed successfully by sequencing and the correct molecular weight of their expression products. Recombinant proteins were obtained in BL21 (DE3) and purified after Ni2+ affinity chromatography. Western Blot analysis and indirect ELISA showed that P2a had specific antigenicity.</p><p><b>CONCLUSION</b>The P2a protein expressed in prokaryotic system was proved to have specific antigenicity. The indirect ELISA distinguished 24 positive sera from 24 negative sera. It is very likely that P2a can be an antigen to diagnose acute patients of hepatitis A and differentiate inactivated vaccine-induced immunity from an infection.</p>

Expression of hepatitis delta antigen Inner Mongolian strain in prokaryotic cell and analysis of its antigenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain high yield and good antigenic activity of HDV L-Ag and to detect different regional patients' sera to test the purified antigen's antigenicity.</p><p><b>METHODS</b>Hepatitis delta virus' sequence was obtained from Inner Mongolian patient by using RT-PCR and PCR methods, PET43a was used and His-tag was added at the HDV L-Ag 5' and 3' to construct the recombinant expression plasmid, transform the plasmid into host bacterium BL21 and induce it with IPTG. The expression supernatant was purified by saturated (NH4)2SO4 and affinity chromatography. The activity and antigenicity of the expressed product were analyzed by using EIA.</p><p><b>RESULTS</b>Comparison of results obtained with detection by using the expressed protein coated plate and ABBOTT Murex anti-Delta (total) of 15 positive and 10 negative sera, the consistency was good (100%).</p><p><b>CONCLUSION</b>EIA proved that the purified antigen had good antigenicity, no serological difference was found in detection between different region's sera, therefore the purified delta antigen may be useful in diagnostic and other research.</p>

Expression and characterization of transcutaneous immunization adjuvant LTB and LTK63 / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study a new kind of adjuvant: transcutaneous immunization adjuvant.</p><p><b>METHODS</b>The full length gene of Heat-labile enterotoxin (LT) was amplified from E. coli H10407. The B subunit protein LTB and the nontoxic A subunit protein LTKA were expressed by genetic engineering manipulation. After purification, they were identified with SDS-PAGE, GM1-ELISA and so on.</p><p><b>RESULTS</b>The LTB protein still persisted its biologic activity that conjugated specifically with GM1 ganglioside, and the LTK63 protein lost its toxin activity.</p><p><b>CONCLUSION</b>The results showed that LTB and LTK63 may be used as promising transcutaneous immunization adjuvant.</p>