Construction Of Phage Screening Model For HIV Protease Inhibitor

One of the two kinds of drugs used in highly active anti-retroviral therapy(HAART) against to acquired immunodeficiency syndrome(AIDS) is protease(PR) inhibitor(PI). While, 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. By analyze the inhibition activity of drug candidates to HIV PR cleaving PR target peptide, the existing PIs drug screening system decide whether drug candidates can become a new PIs. Because different PR target peptide susceptive to different HIV PR, adapted PR target peptides are needed to meet with the resistant mutated HIV PR during the screening of PIs against to resistant mutated HIV PR. Whereas, the most adapted PR target peptides to many kinds of HIV PR resistant mutants are not confirmed. As a result, the existing PIs screening system cannot be used in screening PIs against to resistant mutated HIV PR.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.Three parts are involved in this research: To express, purify and identify the protease of HIV-1 HXB2 subtype in E.coli. , for in vitro research of HIV PR;To cleave target protein CAP2NC and LD3-CAP2NC with fixing tag and CAP2NC protein displayed on the surface of phage with HIV PR in order to establish in vitro model of HIV PR cleaving PR target sequence displayed on the surface of phage;To randomize the HIV PR target site P2/NC sequence and to construct its phage displaying library.1. Expression of Protease of HIV-1 HXB2 Subtype in E.coli and Purification and Identification of Expressed ProductThe 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.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 pQE30 vector which is used as expression vector in E.coli. Expression of HIV PR was induced by IPTG in E.coli Ml 5 and the expressed PR protein waspurified by the Ni-NTA affinity column.The purified PR protein was analyzed by SDS-PAGE and ELISA. 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 pQE30-pr was constructed successfully, The HIV-1 PR was expressed in E.coli Ml 5 after the induction by IPTG with a relative molecular weight of 14000. The purifyed PR protein has a concentration of 7.74mg/ml and showed specific reaction to the anti-HIV positive blood sera.2. Construction of HIV-1 PR target site P2/NC sequence radomizd library and cleavage screening.Construction of conformable large molecule mutation library is pivotal for molecular evolution study in vitro. In order to extend the range of the screening region of amino acid in PR target peptide, PR target peptide was mutated randomly by overlap PCR amplification with the random nucleotide primers, The HIV PR target site P2/NC sequence was randomized and the phage displaying library was constructed. HIV-1 gag CAP2 fragment with induced randomized protease target sequences and NC fragment ware generated by PCR. The CAP2/NC fragment with randomized PR target sequences between CAP2 and NC fragment was generated by overlapping PCR, and cloned into phage display vector pCANTAB5S-LD3. The phage displayed library was generated by M13KO7 rescue. The library was fixed on plates and cleaved by HIV SF2 PR for several times to obtain phages susceptive to PR.As much as 2.1*106 clones were obtained in the phage library and the titer was 3.0x1012TU/ml. About 52.1% clones contained inserted CAP2/NC fragments. Sequence analysis of 12 samples showed that nucleotide acids and amino acids at randomized PR cleavage site distributed randomly. Nine of 10 positive monoclone phages were proved of special binding activity to human IgG Protease target site P2/NC sequences in HIV-1 Gag protein.has been,successfully randomized and displayed on phage surface. The capacity, variety, randomicity and IgG binding activity of the library meet the requirements for in vitro screening the phage with susceptible PR target sequences.After this phage library was fixed and cleaved by HIV SF2 PR, no phage with mutated PR target sequence was obtained as expected after repetition for several times.3. Establishment of cleavage model of HIV PR cleaving PR target sequence displayed on the surface of phageAs PR target sequence susceptible to PR has not been obtained after PR target P2/NC sequence randomized library was cleaved by HIV-1 PR, we, attempted to findthe causes in the following ways: 1, whether HIV-1 PR we used is active in cleavage and whether the PR target sequence can still be cleaved by HIV-1 PR after infused with fixing tag. 2, Whether PR target sequence displaying on the surface of phage can be cleaved by HIV-1 PR. 3, If the cleavage of HIV-1 PR to PR target sequence displayed by phage is viable, whether this cleavage can be checked out effectively. So following works were processed.To confirm the cleavage activity of HIV-1 PR and the effect of LD3 on the cleavge, which is used to fix the phage while in cleavage screening of PR target peptide susceptive to PR, we have expressed both the HIV-1 PR target protein CAP2NC and LD3-CAP2NC protein infused with LD3. The CAP2 DNA and NC DNA was amplified by PCR .The CAP2NC DNA was cloned into pCANTAB5S-LD3 phagemid after amplification by overlapping PCR. The CAP2NC DNA was successfully synthesized and its corresponding amino acid sequence is identical with the primary designed amino acid sequence. The CAP2NC DNA and LD3-CAP2NC DNA were both amplified and added with restrict enzyme site, then cloned into pQE30. The CAP2NC protein and LD3-CAP2NC infused protein were cleaved by HIV PR after being expressed and purified successfully. The cleave result show that the HIV CAP2NC and LD3-CAP2NC infused protein can be cleaved by HIV PR effectively. As a result of infused with LD3, the LD3-CAP2NC infused protein was cleaved by HIV PR less efficiently than CAP2NC protein.To confirm whether the HIV PR target peptide displayed on the surface of phage can be cleaved by HIV PR efficiently, the phage pCANTAB5S-LD3-CAP2NC displaying HIV-1 PR target site P2/NC sequence were prepared and the titer reached to 3.0xl012TU/ml. After fixed on the plates, the phage was cleaved by HIV PR. The result analysed by HRP/Anti-M13 conjugate show that the HIV PR target peptide displayed on the surface of phage can be cleaved by HIV PR efficiently and the cleavage can be checked out by ELISA effectively. The model of HIV PR cleaving PR target sequence displayed on the surface of phage was established successfully.To confirm the validity of this model, one of the Pis Indinavir was used and it showed effective inhibition to the HIV PR cleavage, which confirmed the specific effect of cleavage of HIV-1 PR to PR target sequence displayed by phage. Furthermore, this model of cleavage of HIV-1 PR to PR target sequence displayed by phage base the model of screenig Pis.So we can conclude that: HIV-1 PR used in our research has favorable activity in cleavage on PR target both in vitro and on the surface of phage , and the cleavage can be identified by ELISA effectively. While, the unspecific cleavage was existing between HIV-1 SF2 PR and pCANTAB5S-LD3 phage, which maybe just the cause of none positive result was obtained in the second part. So we are engaged in search for more adapted fixing tag in order to reconstruct the PR target sequence randomized library for screening PR target sequence susceptible to PR and build phage model of screening for Pis.In this work, we succeeded in displaying the HIV-1 CAP2NC protein within the HIV PR target site P2/NC sequence on the surface of phage and the phage was successfully cleaved by HIV SF2 PR. The model of HIV PR cleaving PR target sequence displayed on the surface of phage was established successfully, which confirm the feasibility of screening for PR target peptide high susceptivity to PR cleavge. As the cleavge model can reflect the inhibition of Pis to HIV PR preferably, this model can also be used as a primary screening system for Pis. Furthermore, the model of protease cleaving the protease target peptide displayed on the surface of phage provides a feasible platform for studying the interaction between enzyme and substrate.