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Construction of HIV-1 Tat Core and Basic Region Mutation Libraries and Affinity Screening

Author: PangQiang
Tutor: PanWei；QiZhongTian；CaoJie
School: Second Military Medical University
Course: Microbiology
Keywords: HIV-1 Tat Core basic region Random mutations Phage display And screening pro-
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 52
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Abstract

Human immunodeficiency virus (Human immunodeficiency virus, HIV) attributable to Retroviridae in lentiviruses, mainly through blood, sexual contact and mother to child transmission lines, etc., can cause acquired immune deficiency syndrome (Acquired Immune Deficiency Syndrome, AIDS), namely HIV. Currently, HIV infection has become the world are facing a major public health problem. Developing an effective HIV vaccine is an important means of AIDS prevention. Therefore, to explore new strategies for HIV vaccine research and finding new vaccine targets successfully developed the vaccine is particularly important. HIV-1 trans-activator protein Trans-activator of transcription, Tat) is produced early in HIV infection is an important regulatory protein in the HIV-1 replication, proliferation and play an important role in pathogenesis. HIV-1 infected target cells, synthesized in the cytoplasm to the nucleus transferred Tat, with a variety of transcription factor binding transcription initiation complex composition (transcription initiation complex, TIC), to promote the transcription of viral RNA, extension and viral replication . In addition, Tat by infected cells may also be secreted into the extracellular many ways to play \/ STAT signal transduction via activation of HHV-8 replication, or by RGD (Arg-Gly-Asp) and endothelial cell αvβ3 and α5β1 integrin binding, and soluble inflammatory cytokines and angiogenic factors synergistically promote Kaposi's sarcoma (Kaposi's sarcoma , KS) formation; ③ calcium flow by affecting nerve cells and induce macrophages and microglial cells expressing TGF-β, TNF and other neurotoxic substances, play a neurotoxic role in promoting AIDS encephalopathy Tat basic amino acid enrichment District (49-57aa) is Tat wear membranes and nuclear localization functions motifs Tat protein is the major neutralizing epitopes world. Tat antibodies which eliminate the penetration of molecules other cells play a specific toxic core functions. In addition, Tat basic region sequences are highly conserved in all subtypes, is conducive to a broader spectrum of cross-reactive antibodies. Studies have shown that building Tat51 bit, 55 site-directed mutagenesis, the basic region epitope unspoiled and Tat penetrating activity and extracellular activity was greatly reduced. Therefore, this study intends to molecular evolution using phage display technology platform Tat basic region for natural transformation and restructuring screening Tat basic region in order to ensure the integrity of the epitope, we reserve the Tat core area (38-48aa), constructed by the mutations in the basic region of the core connecting peptide fragments were randomly repeating series of interconnected phage display library containing secondary and active Tat antibody rabbit antiserum by affinity screened to obtain a series of the basic region of the epitope conformation stability of the biological activity was significantly reduced in the mutant sequence, Tat vaccine development for the new do basic research. This study consists of the following three parts: one, HIV-1 PET32a-Tat38-61 protein expression and immunoreactivity identified functional studies show that: Tat protein core area (38-48aa) with uninfected bystander cells binding to tubulin induced apoptosis; Tat protein basic region (49-57aa) Tat protein-mediated trans-membrane, and extracellular Tat activity are closely related. Structural studies show that: Tat molecule is a natural non-folded protein belonging to random coil types of molecules, lack of stable secondary structure and higher order structure, it is very unstable molecule conception, for dynamically changing rapidly, as a result in the Tat antigen stimulation is poor, high-affinity antibodies, and less likely to be degraded. Therefore, Tat basic region important core epitope are able to get a reservation is mutated phage library and screening pro basis. To this end, we first Tat core basic region 38-61aa built into the prokaryotic expression vector, purified PET32a-Tat38-61 protein, and by containing Tat antibody positive serum immunoreactivity its identification. According to E. coli preference codons optimized type HXB2 strains of HIV-1 Tat DNA primers were designed, synthesized by PCR HIV-1 tat38-61 series, T / A cloning and cloned into pMD18-T vector and sequenced. The correct sequencing tat38-61 was cloned into the prokaryotic expression vector pET32a, prokaryotic expression plasmid pET32a-tat38-61. The expression plasmids were transformed into E.coli BL21 (DE3) in, IPTG induced expression of the relative molecular weight of 21,300 fusion protein with Ni-NTA affinity chromatography PET32a-Tat38-61 protein. Were prepared in our laboratory using rabbit anti-PEPTIDE-Tat1-101 serum and Shanghai Public Health Center provides HIV-positive serum ELISA detection, identification PET32a-Tat38-61 protein immunoreactivity. The results showed that: ① PET32a-Tat38-61 with rabbit anti-PEPTIDE-Tat1-101 serum showed specific reaction, and the reaction strength is lower than the positive control protein PET32a-Tatl-101, a carrier protein PET32a a weak response. ② In the 12 cases of HIV-positive serum, 4 patients with PET32a-Tat38-61 has a clear specific response in 6 cases and the positive control full-length protein PET32a-Tat1-101 showed a specific reaction, and contains all the four cases with PET32a-Tat38-61 showed a specific response to HIV serum. ELISA assay described above results PET32a-Tat38-61 protein is better to retain the Tat basic region epitope. In summary, we have successfully constructed and expressed PET32a-Tat38-61 protein was identified by ELISA immunoreactivity showed that with Tat antibodies in serum and PET32a-Tat38-61 fusion protein was specifically react, indicating PET32a- Tat38-61 protein epitopes important basic region be retained for further preparation for the core basic region of neutralizing antibodies and random mutation and screening pro-library basis. Two, HIV-1 Tat basic region of the core combinatorial library of random mutagenesis studies have shown that two important basic region loci 51K and 55R mutagenesis after its epitope without damage, and the activity of Tat transmembrane and extracellular activity was greatly reduced. Therefore, to obtain a series of basic region epitope conformation stability of the biological activity of the mutants was significantly decreased, we first constructed for the basic region of Tat mutant libraries. Containing a nucleotide sequence by using a random primer, Overlap PCR method 51K basic region and two loci 55R a random mutation into the 51X and the 551X. In addition, the building is suitable for large-capacity mutant library of biological macromolecules in vitro molecular evolution is the key. Therefore, in order to expand alkaline region gene sequences were screened range, while in the alkaline zone 61 after the introduction of six randomly connecting peptide X6, in order to expand the library of recombinants, and connect using random peptide epitopes with the basic region interaction between sequences, showing stable conformational epitope. After four rounds of PCR to obtain tat38-61 (51X/55X) mutant fragment was digested by Xba Ⅰ, pCANTAB5S cloned into phage display vector and transformed into competent TG1 cells super established HIV-1 Tat core alkaline region (TCBR) combinatorial library of random mutations. M13K07 helper phage rescue, got TCBR random mutation phage display library primary. The resulting phage display library storage capacity of 5.0 × 106, a titer of 2.65 × 1012 TU / ml, 70% fragments were inserted. TCBR of 24 combinatorial library of random mutation recombinants were sequenced, the results show the mutant library fragments important point mutations 51, 55 two loci with the amino acid sequence of the nucleotide sequence showed a random arrangement, no significant bias. In summary, we successfully constructed the core HIV-1 Tat basic region combinatorial library of random mutations, and its storage capacity, diversity, library construction randomness meet requirements for high school and active follow-containing anti-Tat rabbit antiserum screened by affinity and laid the foundation. Three, HIV-1 Tat basic region of the core combinatorial library of random mutation and screening of affinity by the first part of the identification, we found that the basic region could better retention peptide epitopes which we in two rabbit antisera: Rabbit anti PET32a-Tat1-101 and rabbit anti PET32a-Tat38-101, while the TCBR random mutation and screening combinatorial libraries for affinity, in order to obtain a stronger affinity, less penetrating activity novel immunogens representative sequences. There are two rabbit antiserum simultaneously conduct two library screening, after the end of each round, picked clones were identified by PCR and PCR positive clones were identified through sequence analysis. Two kinds of Tat rabbit antiserum against TCBR library two recombinants were screened by PCR results showed that: ① TCBR fragment insertion mutant library rate rose 70 percent to 90 percent, indicating that Tat antibodies with high affinity mutant fragments by screening effective enrichment; ② fragment containing two mutations in the library recombinants proportion decreased significantly, while containing a single mutation in the recombinant fragment library screening before by the proportion of 55% to 90% of . Theoretically, by connecting a plurality of tandem duplication mutant peptide formed large polypeptide fragments, as affinity relationship epitope selective advantage repeated more times, it is easy to evolve screened. But two screening mutant fragment structure changes, seem to indicate an important fragment of mutant mutation sites 51, 55 of the mutations on the advantages of larger molecules epitope peptide enrichment has a more important role. Two kinds of Tat rabbit antiserum against TCBR library after each round of screening, on 40 recombinants were sequenced, the results showed: ① 51 位 basic amino acids are enriched, which agrees with the theory of natural Tat 51 bit lysine (K ); ② 51-Bit, 55 have appeared characteristic amino acid proline (P). A Japanese experimental results seem to suggest that mutations into the P-51 is more conducive to Tat trans-membrane, which is the opposite of the purpose of the experiment, but the emergence of the same characteristics as amino acids, this link also needs further study. Structure itself seems easier proline stable conformation epitopes. ③ 51S-55P can be characterized as a meaningful candidate sequences for further study. Otherwise a French experimental results show that the Tat basic region of 51 lysine (K) mutagenesis threonine (T), 55 Arg (R) site-directed mutagenesis to leucine (L ) constituting mutant \According to classification of amino acids, 51 T is a polar amino acid, and the filtering characteristic obtained by the same amino acid S is also a polar amino acid, L is a hydrophobic 55 amino acids, and the filtering characteristic obtained by the same amino acid P and also hydrophobic amino acids, therefore, according to the selected characteristic amino acids combined with the existing studies can be 51S-55P features as a meaningful candidate sequences for further study. In summary, we have succeeded in two rabbit antiserum against TCBR random mutagenesis library were two affinity screening, and candidate recombinants by sequencing analysis, we found an important locus mutation fragments 51, 55 appearance characteristic amino acid, 51X for the P \\ S, 55X for the Y / P, where, 51S-55P can be used as a meaningful sequence candidate feature optional further study. This topic was constructed pET32a-tat38-61 prokaryotic expression vector, purified and identified PET32a-Tat38-61 protein immunoreactivity for the subsequent use of rabbit antiserum against Tat basic region mutant library screening foundation; successfully constructed. HIV-1 Tat core basic region random (TCBR) combinatorial library of mutants and in two anti-Tat rabbit antiserum PET32a-Tat1-101 and PET32a-Tat38-101 carried out two clone library screening and found that mutation screening recombinant Fragment important sites 51, 55 amino acids characteristic appearance, 51X for the P \\ S, 55X for the Y / P, where, 51S-55P can be characterized as a meaningful candidate sequences further study. This study attempts to apply to phage display technology-based means of molecular evolution of HIV-1 Tat basic region 株 transformation and screening, in order to obtain a series of basic region epitope conformation and stability, while the biological activity was significantly reduced mutant sequence, Tat vaccine development for the new to do basic research.

Construction of HIV-1 Tat Core and Basic Region Mutation Libraries and Affinity Screening

Abstract

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