Clone, Expression, Purification, Identification And Primary Application Of HIV-1 Gp120 Gene Fragment

Objective: Human Immunodeficiency Virus (HIV) is the cause of Acquired Immunodeficiency Syndrome (AIDS). HIV was devided into two types, HIV-1 and HIV-2. HIV-1 has disseminated broadly all over the world and it is the main cause of AIDS. HIV envelop antigen gp120 (HIV-1 env gp120, gp120) is one of the main antigen of HIV-1, it is important material in HIV detection kit for anti-gp120 antibody and it's the necessary material in preparing gp120 antibody. The gp120 protein was used broadly and of great significance. The present study is intended to obtain highly purified protein of gp120 by gene engineering.Methods: The gp120 gene fragment was amplified by PCR from the HIV-1 HXB2 cDNA template and linked with T vector, pMD-18T, for constructing the recombinant clone plasmid pMD-18T-120. The recombinant clone plasmid was identified by PCR, restriction endonuclease analysis and DNA sequence analysis. The correct recombined plasmid was then digested by NdeI and XhoI. The recombinant expression plasmid pET-22b-gp120 wasconstructed by inserting the obtained gene into the MCS of plasmid pET-22b. The recombinant expressing plasmid, pET-22b-120, was identified by PCR, restriction endonuclease analysis and DNA sequence analysis, then transformed into E.coli. BL21(DE3). A small number of transformed E.coli. BL21(DE3) was induced by IPTG. The thalli was analyzed by SDS-PAGE for selecting the positive clone that can express the intended protein. The selected clone was amplified in 2 Liter LB culture medium and induced by IPTG for 4 hours. The thalli was selected and ultrasonicated. The inclusion body and the supernatant were separated. It can be approved by SDS-PAGE that the target protein was mainly indwelled in the inclusion body. The inclusion body was washed and then lysed by Guanidine HCl. Purified gp120 was obtained through Metal Chelate Affinity System and Gel Filtration, detected by Western blot and ELISA. The molecular weight and purity of the purified gp120 were analyzed by SDS-PAGE and HPLC, respectively. Using the purified gp120 antigen as an antigen, a serum gp120 antibody detection kit based indirect ELISA methods was constructed. The sensitivity and specificity of the kit was determined by detecting 10 international standard HIV sera. Results:1 Detected with agarose gel electrophoresis, an apparent stripe in the PCR product between the 500 bp and750 bp of DL2000 marker was identified, which coincide with the anticipation of 667 bp of HIV-1 gp120 gene fragment anticipated. 2 By agarose gel electrophoresis of products of PCR and restriction endonuclease analysis for the recombinant clone pasmid pMD-18T-120, an apparent strip appeared between the 500 bp and 750 bp of DL2000 marker, which coincided with the 667 bp of HIV1 gp120 gene fragment anticipated. The fragment was coincided with the anticipated HIV gp120 by DNA sequencing. The above result indicated that the intended gene was correctly cloned.3 By agarose gel electrophoresis of products of PCR and the result of restriction endonuclease analysis for the recombinant expression plasmid pET-22b-gp120, an apparent stripe appeared between the 500bp and the 750bp of DL 2000 marker, which coincided with the 667bp of HIV-1 gp120 gene fragment anticipated. DNA sequencing demonstrated that the fragment was coincided with the anticipated HIV-1 gp120. All these indicated that the recombinant expression plasmid was constructed successfully. 4 SDS-PAGE analysis showed that there was an apparent stripe between 21.1KD and 30 KD. The stripe was the product of E.coli transformed by the recombinant expression plasmid pET-22b-gp120 and induced by IPTG,and its molecular weight was coincided with the 25kD of protein anticipated. The recombinant protein was mainly dwelling in the inclusion body and it took 24.72% of the total thalli protein, 57.85% of the inclusion body by gel scan image analysis.5 HPLC analysis showed that the purity of intended protein purified by Ni2+ affinity chromatography and gel chromatography was 95.