Gene Cloning And Expression And Serodiagnostic Value Of Herpes Simplex Virus Type 2 Glycoprotein G-2 Unique Domain

Significance and ObjectiveHerpes simplex virus (HSV) belongs to Herpesviridae a subtype, its plasmid is 180 nanometers. According to antigenic difference, the virus has type land type 2. HSV infection is widespread in the crowd, because people are only physio-host of herpes simplex virus. Herpes simplex virus type 2 mainly concerned with infection of anoperineogenital and newborn, and it can induce genital herpes and newborn infant herpes. By medical science study for many years, HSV infection is related with uterine cervix cancer in woman patients, and increases chance of AIDS infection. Disease incidence of genital herpes is second only to gonorrhea and syphilis, occupying NO. 3, and is the first in sexually transmitted disease caused by virus. Genital herpes clinical manifestation is multiformity, it possibly is typical blister and anabrosis, but non-typical and subclinical infection is more visible which is easily ignored by people. Therefore today medical science workers think highly of the genital herpes again. Now, Test facility of herpes simplex virus infection is limited, for example that complement fixation of serology classic method can not identify HSV-1 and HSV-2. Orthodox virus cultivation is time-consuming and exhausted. So in our study we aim at founding Herpeselect HSV-2 enzyme-linked immunosorbent assay (ELISA) and Western blot on account of type-specific HSV-2 glycoprotein G to detect HSV serum antibody, and these methods will be fast and effective diagnostic method.Many study and researches manifest that HSV viral particle has at least 12 glycoprotein, these are gB,gC,gD,gE/gI,gG,gH/gL,gK,gM/gN,gJ. Among the total, gG take along type-specific antigen determinant, gG-2 takes a big role in diagnosis, typing and vaccine study of herpes simplex virus type 2. US4 encode gG-2, and gG-2 gene fragment length is about 2100bp, can encode 699 amino acids. We analyze B cell antigen epitope between gG-1 and gG-2 of HSV, and compare their gene difference. Gene total length of HSV-2 gG-2 has high congelleric domains with HSV-1 gG-1 and unique domain. There is very low homology in unique domain between gG-1 gene and gG-2 gene. There are 7 antigen index number and surface possible fortis peptide section relative concentrate district. Other researchers find that gene antigen district of HSV-2 gG-2 happen mutation seldom. Even if gene mutation appears, that also can not weaken serology activity. They also confirm that gG-2 gene can be good type-specific antigen, and gG-2 gene's conservatism is prerequisite of HSV-2 idio-vaccine, thus, we make unique domain of gG-2 gene as target protein antigen, amplifying and cloning it, then expressing it in prokaryotic system and cleansing, at last, we get antigen activity of interest protein studied initially.Method and Content1,Drawing and separating the materials from blister, which patients are clinical definite as HSV-2, and cultivating virus in Vero cell.2,According to genome series of HSV-2 glycoprotein G provided from GenBank, to design amplification primer, the upper stream primer series is "5'- GAATTCatggcacgacccacggaagacg -3'"; The down stream primer series is 5'- CTCGAG cggggttgcgggtccgg -3'. Making DNA extracted from supernatant of virus culture fluid, we amplify gG-2 gene and construct cloning vector pGEM-T-G2, utilizing PCR to identify it.3,Rrecording HSV-1 glycoprotein G-1 series from GenBank to input Editseq software of the LaserGene package. Cutting gG-1 gene fragment and translating it into amino acid sequence. To utilize Clustal X software, we compare nucleotide and amino acid sequence between amplified gG-2 gene and cut gG-2 gene. To utilize Proetean and DNAMAN software, we also make a forecast about epitope,αcoiling center and hydrophobic region, and compare difference of them.4,According to sequence analysis results, we select unique domain (gG-2_T) of gG-2 gene as purpose order. On the basis of gene order of herpes simplex virus 333 stock, we design its primer and add EcoRⅠ/XhoⅠrestriction enzyme situs in both side primer. We make pGEM-T-G2 plasmid formwork to amplify gG-2_T, getting pGEM-T-gG-2_T recombinant cloning vector.5,Connecting pGEX-4T-1 carrier and pGEM-T-gG-2_T after they are both enz-cut by EcoRⅠ/XhoⅠto construct fusion expression vector pGEX-4T-1-gG-2_T to convert it into parasitifer BL21. By enz-cutting and PCR, they will be accredited and sequenced.6,We induce to express pGEX-4T-1-gG-2_T by utilizing IPTG and initially accredit express protein by SDS-PAGE electrophoresis and Western blot.7,We optimize expression condition and express interest protein in supernatant and purify expression protein by using GST affinity purification kit.8,We make Western-blot and indirect ELISA by utilizing pure protein to respectively detect serums of HSV-1, HSV-2 and health adult.Finding1,Vero cell degeneration, the virus amplify in Vero cell successfully.2,Extracting virus DNA, amplifying herpes simplex virus type 2 gG-2 gene, gG-2 gene length is about 2100bp, the homology is 99.45% between it and gG-2 gene of herpes simplex virus type 2333 stock. Making gG-2 gene as template to amplify and clone gG-2_T, series length of gG-2_T is 591bp, encoding 197 amino acids.3,We synthetically predict glycoprotein G of HSV-1 and HSV-2 according to Poly-Kyte-Doolittl hydrophilicity, Polt-Emini surface possibility and Jameson-Wolf antigen index number by bioinformatics software analysis.4,We optimize expression condition to express pGEX-4T-1-gG-2_T into fusion protein in Escherichia coli BL21, which protein molecular mass is about 46KD. By supersound spallation, fusion protein is dissolved in lysate as supernatant. Fusion protein has GST label, can pass GST affinity purification pillar to be purified.5,By setting condition optimized, we reduce protein polluted effectively. Content of purification protein is GST 0.85mg/ml and HSV2-G-2 unique domain 0.49mg/ml. purity coefficient of interest protein exceeds 95%.6,Interest protein purified take reaction with GST polyclonal antibody, also make reaction with patients serum of herpes simplex virus type 2, but not react with HSV-1 patients serum. So we get interest protein that we need.ConclusionManufacturing new type specificity diagnostic kit and developing vaccine are best effective method to diagnose and prevent herpes simplex virus infection now respecting significance of preventing, detecting and treating HSV infection. The former can diagnose and identify primary infection and reinfection, at the same time can make large scale epidemic screening. The latter can bring into full play humoral immunity and cellular immune function to wipe out HSV infection in reject HSV infection immunity. HSV-2 gG-2 gene fragment length is about 2100bp, can encoded 699 codons by US4, including a unique domain and a height congelleric area with HSV-1 envelope glycoprotein gG-1. By comparing gG-1protein and gG-2 protein and making a forecast to B cell epitope of it, we find there is very low homology in unique domain between gG-1 gene and gG-2 gene. Grabowsk found tow immunizing sites in amino acid residue 351～427,525～587 in gG-2 "unique domain" by bacterial invader technique. He also authenticated the two amino acid residue just react with HSV-2 not HSV-1 in test. Ikoma expressed gG-2 (281aa-594aa) by using granulosis virus expression system, and got interest protein to detect HSV-2 infection patiens serological specific antibody, the specificity and sensitivity is 95.5%. The study illustrated that the tow district peptides are fit for clinical antibody detection, especially fitting for typing diagnosis.A lot of study and research find that gene antigen district of HSV-2 gG-2 happen mutation seldom. Even if gene mutation appears, that also can not weaken serology activity. This report also confirmed that gG-2 gene can be typical specific antigen, and conservatism of gG-2 gene is the prerequisite as HSV-2 specific vaccine.We construct HSV-2 gG-2 unique domain gene fragment (285～482aa) successfully by molecular cloning and gene recombination technology, and express fusion protein included gG-2 unique domain by using prokaryotic expression vector, then detect expression protein can specifically bind with GST polyclonal antibody by Western blot. Purified protein is our needed interest protein. Indirect ELLISA proves expressed protein can take reaction with serum of HSV-2 infection patiens, not r-Globulin and HSV-1 patiens, which manifest prokaryotic expression protein has immunologic competence. Above study provides information for inventing type specific diagnosis kit of HSV-2 infection, genital herpes seroepidemiological survey and type specific vaccine study.