Prokaryotic Expression And Purification Of Protease Of HIV-1 HXB2 Subtype And Identification Of Cleaving Activity

In the post decades,infection of HIV mushroomed in the world, the acquired immunodeficiency syndrome(AIDS) has becamed the most fearful epidemical disease which jeopardizes humankind's healthiness worldwide. One of the two kinds of drugs used in highly active anti-retroviral therapy(HAART) against to AIDS is protease(PR) inhibitor(PI) integrates two reverse transcriptase(RT) inhibitor. however, the emergence of mutated PR of human immunodeficiency virus(HIV) has blocked the antiviral therapy. Therefore, more and better PIs are needed for clinical usage. at the same time, all of circs require new powerful screening model to proof-test new drug,which could work with existent screening models complementary.So we attempt to cleave the phage library displaying PR target peptide randomized mutants by resistant mutated HIV PR, in order to select PR target peptide with high susceptivity to resistant mutated HIV PR cleavage. These PR target peptides high susceptive to resistant mutated HIV PR can be used to screening PIs against to resistant mutated HIV PR. Screening system of PIs against to resistant mutated HIV PR can also be established on the base of phage displaying PR target peptides high susceptive to resistant mutated HIV PR and resistant mutated HIV PR. The expression of HIV PR is a premise to screen susceptive PR target peptide, so we selected HIV-1 HXB2 PR for expression, purification and identification, which is the representative of HIV-1 M group B clade. Three parts are involved in this research:1. To express and purify the protease of HIV-1 HXB2 subtype in E.coli. ; 2.expressing target protein CAP2NC of protease to check up the cleaving activity of protease; 3. refold PR and cleave the target protein CAP2NC; 4. cleave the model of HIV PR cleaving PR target sequence of CAP2/NC displayed on the surface of phage. 1. To express and purify the protease of HIV-1 HXB2 subtype in E.coli. .The primers were designed according to the PR amino acid sequence of HIV-1 HXB2 subtype and the E.coli preferred codon. The PR DNA fragment with E.coli preferred codon was synthesized in vitro by overlapping PCR and sequenced after inserted into pMD18-T vector by T/A method. Then the PR DNA sequence was cloned into pET-32a vector which is used as expression vector in E.coli. Expression of HIV PR was induced by IPTG in E.coli BL21 and the expressed PR protein was purified by the Ni-NTA affinity column.The purified PR protein was analyzed by SDS-PAGE. The HIV PR DNA fragment with E.coli preferred codon was successfully synthesized and its corresponding amino acid sequence is identical with the primary amino acid sequence of HIV-1 HXB2 subtype. The expression vector pET-32a -pr was constructed successfully, The HIV-1 PR was expressed in E.coli BL21 after the induction by IPTG with a relative molecular weight of 30000. The purifyed PR protein has a concentration of 2.54mg/ml.2. Expressing target protein CAP2NC of protease to check up the cleaving activity of proteaseThe primers were designed according to the CAP2NC amino acid sequence of HIV-1 HXB2 subtype and the E.coli preferred codon. The CAP2NC DNA fragment with E.coli preferred codon was synthesized in vitro by overlapping PCR and sequenced after inserted into pMD18-T vector by T/A method. Then the CAP2NC DNA sequence was cloned into pQE30 vector which is used as expression vector in E.coli. Expression of HIV CAP2NC was induced by IPTG in E.coli M15 and the expressed CAP2NC protein was purified by the Ni-NTA affinity column.The purified PR protein was analyzed by SDS-PAGE. The HIV-1 CAP2NC was expressed in E.coli M15 after the induction by IPTG with a relative molecular weight of 35000. The purifyed CAP2NC protein has a concentration of 4.50mg/ml.3. Refolding PR and cleave the target protein CAP2NC to test PR's cleave activityWe attempt the dilution method,dialysis method,pulse dilution method and make use of PBS refolding buffer(0.01M PBS ,10% glycerol ,2mM DTT ,pH7.0),NaAc refolding buffer(50 mM NaAc, 10% glycerol, 2mM DTT ,pH5.2),MES refolding buffer( 20mmol/LMES ,2 mmol/L DTT,10% glycerol,PH 5.5),Tris·HCl refolding buffer(0.01M Tris,10% glycerol, 2mM DTT, pH5.2)in the work of refolding PR,the result show that use MES refolding buffer( 20mmol/L MES ,2 mmol/L DTT,10% glycerol,PH 5.5)and dilution method get the best refolding effect,the refolded PR can cleave substrate protein CAP2NC and the cleave activity can be restrain by Indinavir.4. The experimentation of HIV PR cleaving PR target sequence CAP2/NC displayed on the surface of phageWe have succeed constuction the phage library of display randomization sites on CAP2/NC sequence of HIV-1 PR target sequence, in this work ,we inoculate the phage library to E.coli TG1 and dilute the titer of second phage library to 1012TU/ml, The phage vector pCANTAB5S-F7 was able to bind to IgG validly. We fix IgG on solid surface, adds HIV PR to cleave the phage libraries, the result show that the HIV-1 PR we expressed can not cleave the phage library of display randomization sites on CAP2/NC sequence of HIV-1 PR target sequence.In this work,we we succeeded prokaryotic expression and purification of Protease of HIV-1 HXB2 Subtype and PR's substrate protein CAP2NC, the refolded PR can cleave substrate protein CAP2NC and the cleave activity can be restrain by Indinavir. The PR we expressed can make use of the cleaving screenning of the HIV-1 Gag CAP2/NC protein phage displayed library with randomized P2/NC protease cleavage site and establish a phage model for in vitro screenning of PR inhibitors.