The Expression Of Recombinant Antigen Env Of Htlv-â… 

Background and Objective:Human T-cell Lymphotropic Virus(HTLV) is the first discoveried virus among human retrovirus.It belongs to Retroviridae,Oncovirinae,mammalian C-type virus. HTLV hasⅠtype andⅡtype.HTLV-Ⅰcan lead to adult T cell leukemia/lymphoma (ATL) and Tropical spastic paraparesis/ human T-cell lymphotropic virus typeⅠ-associated myelopathy(TSP/HAM) et al.HTLV-Ⅱmaybe relate to T cell hyperplasia disorder et al.HTLV-Ⅰis prevalence in the world,and its incidence rate are high in the local regions.The infective cases of HTLV have been found in more than 10 provinces of China,and been prevalent intensively in some littoral area,such as Fujian province and Guangdong province.HTLV was mainly transmited by blood transfusion,sexual contact and lactation.It has been classified as one of requisite checking items to blood donators in Japan,USA,Australia,France and many other countries.Now,all of domestic HTLV screening reagents need to be imported from other countries.And HTLV-Ⅰspecific antigen always hold back the development of domestic HTLV screening reagents.In our study,recombinant antigen env had been expressed by prokaryotic expression vector and eukaryotic expression vector through technique of molecular biology,which can provide useful materials for the development of domestic HTLV screening reagents.Methods:After HTLV-Ⅰenv gene and the amino acid sequences of its gp46 protein and gp21 protein was analyzed,a 630bp gene sequence(5915nt-6545nt) which has abundant antigenic determinants was selected as target gene.And the target gene were synthesized artificially,which would be added two restriction enzymes(BamHⅠand PstⅠ) sequence in both sides.Then the target gene were respectively cloned into prokaryotic expression vector pQE80L and eukaryotic expression vector pcDNA4/HisMax-A.The positive recombinants(pQE80L-env,pcDNA4/HisMax-A-env) which were identified by PCR and restriction enzyme digestion analysis was sequenced.Then pQE80L-env was transformed into DH5α, and lmmol/L IPTG was used to induce the host bacteria to express the recombinant proteantigen env which would be purified by affinity chromatography. pcDNA4/HisMax-A-env was transfected into NIH3T3 cell,which were cultured for 48h,and then recombinant antigen env were purified by affinity chromatography.The recombinant proteantigen env expressed in prokaryotic cells and eukaryotic cells were detected by Western-blot.The specificity of two recombinant proteantigens env were detected by ELISA.Results:1.The positive recombinants(pQE80L-env,pcDNA4/HisMax-A-env) were identified by PCR and restriction enzyme(BamHⅠand PstⅠ) digestion analysis. And after sequenced,the target sequences were coincident completely with the design one.2.An expected recombination protein(25KD) was detected in SDS-PAGE analysis. And western-blot analysis shows there was a obvious and specific strap at 25KD position,which proved the prokaryotic recombination protein has the antigenicity of HTLV-Ⅰenv.3.After the NIH3T3 cells were transfected for 48h,the expected recombination protein(25KD) in the culture supernatant was was detected in SDS-PAGE analysis. And western-blot analysis shows there was a obvious and specific strap at 25KD position,which proved the eukaryotic recombination protein also has the antigenicity of HTLV-Ⅰenv.4.The results of recombination antigen env in the prokaryotic expression by ELISA: the reference serum of health adults,HIV(+) sample and HTLV-Ⅱ(+) sample were all negative;the reference serum of HTLV-Ⅰ(+) sample were clearly positive.5.The statistics data show that there were no significant difference between the result of ELISA test on the recombination antigen env expressed by prokaryotic cells and by eukaryotic cells(P＞0.05).Conclusion:HTLV-Ⅰenv gene(5915nt-6545nt) can be used to express the specific recombination antigen env by the prokaryotic expression system and eukaryotic expression system;and the two recombination antigen were no significance on the antigenicity,both of them may be used as the antigen of detection reagent.