Expression Of Anti-HIV-1 ScFv And Construction Of Anti-HIV-1 Immunotoxin

Antibody has been increasingly think highly of as be used for diagnosis and therapy . And following the development of modern genetic engineering , micro-molecular antibodis such as single-chain antibody(ScAb) stept into our sight. ScAb has the superiority especially in the construction of targeting-drug . To obtain no poisonou and no subpoisonou anti-HIV drug , rIT was expressed in E.coli, hope that the rIT's specially selective killing activity can ultimately realize the mind of anti-virus therapy in anti-HIV .To obtain active ScFv product and try to secrete HIV gp41 glycoprotein , two recombinant plasmid pPIC9-ScFv and pHIL-gp41 of Pichia yeast shuttle vector were constructed . First use EcoRI and NotI cut ScFv gene from plasmid pET28-ScFv , then ligate this gene into pPIC9 plasmid cut by the same enzyme . Second use PCR amplify gp41 gene from plasmid pJenlO which contain HIV Env total gene , two EcoRI site were plused in the primers , PCR product cut by EcoRI and ligated into plasmid pHIL-S 1 use T4 ligase . The two recombinant plasmid of Pichia yeast shuttle vectors were lined by BgllII and transformed into GS115 by electroporation . After screened recombinant yeast strain , induce the recombinant yeast secret recombinant proteins by methanol . The secreted ScFv can binding HIV gp120 protein , and secreted gp41 successfully .A rIT fusion gene that using the anti-gp120 ScFv substitutes the receptor binding region of DT toxin was constructed by PCR . Four primers were synthesized , using the DT primers amplify DT gene from plasmid pDT which contain DT toxin total gene ; using ScFv primers amplify ScFv gene plus a peptide connector sequence(ASGGPE) , put the two PCR product together as thumb , use DT front primer and ScFv reveres primer amplify the fusion gene .Cut the PCR fusion gene by Ncol HindIII and ligate the cut gene into plasmid pET28 which cut by the same enzymes . The rIT was expressed in E.coli BL21 strain induced by IPTG , and to advance secretion the recombinant rIT from E.coli in order to check it's activity of killing HIV infected cells .