Constructing A Model Of Screening HIV Protease Inhibitor In Vitro By Phage Display Techniques

Nowadays, we are faced with the difficult challenge to battle with acquired immunodeficiency syndrome (AIDS). It is a serious global puzzle of public health and society.Clinical data showed that the highly active anti-retroviral therapy (HAART), which aims to AIDS, could reduce the rates of disease and death significantly. It uses a kind of HIV-1 protease inhibitor (PI). But, the emergence of mutated protease was hindering the application of HAART. More and better PIs are demanded for clinical usage.Different protease target peptide was susceptive to different protease, so during the screening of PIs that against to the resistant mutated protease, adapted protease target peptides should be meet the demands of the resistant mutated protease. Whereas, the most adapted PR target peptides to many kinds of HIV PR resistant mutants are not confirmed. The current screening mode cannot satisfy the demands of facing all manner of resistant mutated protease.We attempt to construct phagemid libraries, which displaying the protease target peptide randomized mutants, in order to find highly sensitive ones that fed on mutated protease. And these highly sensitive protease target peptides should be screened from the libraries by enzyme action of the resistant mutated HIV protease. We choose the CAP2NC sequence; randomize three amino acids of it, and conjunct to the phagemid vector. The protease target sequences were displayed on the surface of phage. The phagemid could be fixed on the surface that IgG were coating on. Three parts:1. Expression, purification and identification for protease of HIV-1 HXB2 subtype in E.coliThe 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 purified PR protein has a concentration of 2.54mg/ml. The protein was then diluted with 20 volumes of refolding buffer and allowed to stand at 4°C for 12 hours.The part of work was completed by Chen Ming.2. Construction of phagemid libraries to display randomization sites on CAP2NC sequence of HIV-1 gag proteinConstruction of high capacity libraries was essential for molecular evolution study in vitro. We designed primers to implement the mutation of protease target peptides.Methods:Introduce nine randomized bases into 3′terminal of CAP2 fragment by PCR, and obtain CAP2NC fragment that contained these randomized sites and had StuⅠrestriction site on 5′terminal and SalⅠrestriction site on 3′terminal by overlapping PCR. Clone the three different CAP2NC fragment into pMD18-T, then into phage display vector F7-pCANTAB5S.The three phagemid libraries contain 9.7×106﹑6.3×106﹑1.2×107 clones respectively. Each of the titer of the three libraries goes beyond 1013TU/mL. The positive clones reach to 50%﹑62.5%﹑41.67% in the libraries. The analysis of sequences shows that the randomicity and variety of these libraries can meet the demand. About 60% clones can bind IgG protein specifically. So, three CAP2NC fragment libraries, each of which contains nine randomized bases, were displayed by pCANTAB5S-F7 phage successfully. The capacity, titer, rate of positive clone, and randomicity could fit the requests of construction a model for in vitro screening the phages that carried susceptible cleavage sites or sequences.3. Establishment of cleavage model of HIV protease and screening workThe phage vector pCANTAB5S-F7 was able to bind to IgG validly. We fix IgG on solid surface, adds HIV SF2 PR to cleave the phage libraries, and hope to obtain significant ones. There are no significant phages initially. So, we do some research to check the activity of the HIV-1 PR we using. Serials of experiments were designed and enforce: we mixed the positive and negative phages in proportion at 1:1, 1:10, 1:100, 1:1000, and 1:10000. At the rate of 1:1000, HIV-1 SF2 PR could recognize and cleave the target peptides effectively. Meanwhile, we adjust and optimize the condition of the enzyme action. Some sensitive target peptides were obtained. The research is carrying on.We randomized nine bases in CAP2NC-- one of the target peptides of HIV-1 protease, connected it into phage vector pCANTAB5S-F7, and construct three phagemid libraries. The capacity, titer, rate of positive clone, and randomicity could fit the requests of construction a model for in vitro screening the phages that carried susceptible cleavage sites or sequences. The screening system was feasible and some sensitive target peptides were obtained from the library. The new screening system is to bring new ideas of screening drugs in vitro and provide a platform of research for co-action of enzymes and their substrates.