The Establishment And Application Of An Immunological Diagnosis Method Based On The Specificity-epitope Of Herpes Simplex Virus Type 1

A few of diseases could be caused by infection of Herpes Simplex Virus(HSV),which was often found in clinic.HSV has 2 type such as HSV-1 and HSV-2.Herpetic keratitis caused by infection of HSV-1 in corneal epithelium,for high morbidity,obduracy of pathogenetic condition,protraction of course of disease,course of disease,which is thought as the top 1 of ceratonosus of impairment and blindness in the world.Genital Herpes caused by HSV-2 infection is one of venereal disease often found in clinic.Previous Study have dementrated that HSV-2 involve in the tumorigenesis of cancer of the cervix and vagina,oral squamous carcinoma.HSV-2 is the pathogen of TORCH syndrome.Foetus infection of HSV-2 from mother in trimester of pregnancy,led to miscarriage,premature birth,dead foetus.So,it's very important that two type HSV can be efficiently discriminated in clinic for diagnosis and treatment of patients.With the study development of genomics and epitope of HSV,some type specificity epitopes of HSV-1 and HSV-2 were understood by investigators.If a kind of differential diagnosis method for HSV-1 and HSV-2 could be established,people can promoted the differential diagnosis.A type-specific epitope of HSV-1 were expressed by DNA recombination and the recombinated expressed protein were used to establish one serological diagnosis for HSV-1 by ELISA.Methods and results:1.Synthetize gG112-127 gene of 2 copy HSV-1 type specificity epitope antigen,recon including 4×,8×,16×,32×gG_112-127were made by recombination technology through isocaudarner.The results of enzyme cleavage identification and sequencing of Ribonucleotide are agree with the anticipation of theory.2.The expression of multicopyre recombinant can be induced by IPTG.The 8×copy recombinant was expressed by 20.5%,mainly as inclusion body.and the interest protein could be purification by 98.05%o through cleaning and ion exchange chromatography. 3.HSV-1 and HSV-2 were transfered to nitrocellulose after SDS-PAGE respectively, serum of rabbit anti-8×gG_112-127as primary antibody.Specific response of antigen-antibody of rabbit 8×gG_112-127serum was found in HSV-1 but not in HSV-2.the recombination protein of 8×gG_112-127 transferred to nitrocellulose film after SDS-PAGE,the serum of rabbit anti-HSV-1 or HSV-2 as primary antibody.Results showed Specific response of antigen-antibody with HSV-1 but no with HSV-2.4.Indirect ELISA were established and improved by means of purified 8×gG_112-127as coating antigen.The optimized concentration of coating is 5ug/ml,the optimized condition is 37℃4h,and then 4℃to stay overnight.The dilution of patients serum is 1:40,the reaction condition is 37℃60min,5%defatted milk powder as confining liquid.By using the above established ELISA,we Examined 35 cases of HSV-1 infected serum and 43 cases of negative serum.The results showed 91.4%of sensitivity,97.6%of specificity,97.0%of positive predictive value,93.3%negative predictive value,accuracy rating 94.5%.By the above method,we examined 6 cases serum and repeated three times.The reproducibility was very high.Compared with the imported ELISA kit,the kit established by present study had 93%consistency in examining 100 patients serum.Conclusion:gG_112-127 is a type-specific epitope of HSV-1.8×gG_112-127 recombination protein was got by present study,possessing characteristics of favourable antigencity and specific immunoreactivty.The established indirect ELISA for examining HSV-1 by recombination protein had high specificity and sensitivity.The present study provided the basis of theory and practice for developing kit of differential diagnosis of HSV infected patients in clinic.