Chinese Strains Of The Env Gene Cloning. Human T Cell Leukemia Virus Type I (htlv-i), Evolutionary Tree Analysis And The Development Of Recombinant Antigen

The human T-cell leukemiallymphotropic virus type I(HTLV-J) belongs to the Qncovirinae subfamily of retroviruses. It causes adult T-cell leukemiallymphoma (ATL), and a chronic neurologic disease, HTLV- 1-associated myelopathy/tropical spastic paraparesis (HIAMITSP). Phylogenetic amalysis of the long terminal repeat (LTR) and env gene of HTLV-I gave a phylogenetic classification of HTLV-I genotypes into five major molecular subtypes~ Cosmopolitan(C), Japanese(J) , West African (WA) , Central African (CA) , and Melanesian (M). In order to understand the major genotype of HTLVI prevalent in China, HTLV-I env gene was amplified by PCR and sequenced from peripheral blood Iymphocyts of six I-ITLV-I carriers in the Putian region of Fujian Province . Homology comparison and phylogenetic analysis was done with env sequences of six isolates and the representative strains of five major HTLV-I subtypes. The results showed that they have more than 90% sequence similarity between themselves and all the six isolated HTLV-I strains were grouped into subtype C.For development of antibody detection reagent for human Tlymphotropic virus (HTLV), a 212 amino acids length gene fragment from the midpiece of gp46 to the N-terminal of gp2l was selected as well as a type specific epitope region of HTLV type II env gene to construct a HTLV 1+11 chimeric gene. This chimeric fragment was inserted into prokaryote expression vector pRSET and expressed inIIII,E.coli. The yield of the target recombinant protein was about 30%. The final purity is about 95% after ammonium sulfate precipitation and electrophoresis elution purification. Detection by Western blotting, this recombinant protein showed strong reaction with four I-ITLV-L positive sera as well as two HTLV-II positive sera, and had no response to four HTLV negative sera. The purified recombinant antigen, ENV 166, was used as a coated antigen to develop an indirect ELISA for the detection of HTLV infection. The results showed that the OD values of 200 negative sera selected random were less than the Cutoff value, meanwhile, that of 10 HIV positive sera were less than the Cutoff, which indicated that the specificity of our indirect ELLSA could amount to 100%. Both 10 HTLV-I sera and 3 HTLV-JI sera could be found positive with this kit, showed the sensitivity of our ELJSA could reach 100% in detected 13 samples. The antigen, ENVJ66, has been put into production of antibody-specific diagnostic kit for HTLV in our country. As a result, we have received a certificate of new medicine II. I think our studies has provided an important tool for the further research of HTLV in China.