The Research On The Construction, Expression, Purification And Antivirals Against HIV-1 Of Recombinated Human TRIM5α Chimera Protein In Vitro

Aquired Immune Deficiency Syndrome(AIDS),which is a collection of symptoms and infections resulting from the specific damage to the immune system in human,is caused by human immunodeficiency virus(HIV).AIDS patients often have an increased risk of developing opportunistic infections and tumors.HIV-1 is the source of 95%HIV infections throughout the world.Since AIDS was first reported in the United States in 1981,there have been more than 2.5 million people died of AIDS throughout the world.In recent years,scientists hopes to solve the problem of AIDS in the direction of the natural anti-HIV molecules from the cells.Researches have shown that,HIV-1 virus can infect many mammals cells,including human cells,but not some monkey cells. Sodroski Group conduct a study on this fact,an anti-HIV-1 proteins was found in primate cells in 2004,TRIM5α,belonging to the tripartite motif(TRIM) protein family, is a cytoplasmic body protein with three domains.Recently a newly discovered function of TRIM5αisolated from Rhesus monkey is the potential restriction of HIV-1 and this exciting discovery has attracted widespread atention in the world.But so far,the mechanism of TRIM5αrestriction of HIV-1 replication is not yet entirely clear,many questions yet to be resolved,most of them stay at that level.What is the reason for bias of the different species of anti-virus of different types of TRIM5a?Although human's TRIM5a also has the same function,but not the rhesus monkeys. However,when several amino acids change,the human TRIM5 alpha will have similar level of anti-HIV-1 as the TRIM5a rh.Early clinical trials prove that after heterologous protein used in the human body,the immune system can lead to the immune rejection of heterologous proteins,repeated use even can cause severe anaphylactic shock.In addition,because of the immune response of human anti- Heterogeneous animal. heterologous protein in the human body is often quick to be removed,and the half-life is shorter.Therefore our laboratory choose TRIM5α(mutant) come from Homo sapiens to research.But in the past,the majority of the researches about TRIM5αwere finished by gene transfection.For example,TRIM5αgene was conveyed into the cells by liposome or viral vector.But there are many problems during the operative procedure.Such as poor feasibility,low efficiency and stability,low transfection rate,biological toxicity.So at present,the researches about TRIM5αare executed only in cells.the application of the important gene-TRIM5αis restricted.If we can get quantity of active protein TRIM5αin vitro,meanwhile,if the protein can permeate plasma membrane spontaneously,the application range of TRIM5αwill be expanded greatly.In the recent years,people found a kind of protein functional domain,they can guide the biomacromolecule to permeate the plasma membrane and accumulate in the cells,so they were called protein transduction domain(PTD).Those molecules carried into the cells hold their native activity.They can do their native work.So PTD is very charming because the character.It is possible for the macromolecule protein or polypeptide to permeate cytoplasmic membrane because of the PTD,meanwhile,another therapy platform that is to say treatment technic by protein becomes ture.PTD accelerates the protein engineering and the exploitation of protein medicine.At present, many kinds of protein have been carried into the different cells or the animals,they hold their native activity.PTD will be applicated to find new therapy method and new function of protein.But in vitro,does the fusion protein TRIM5αhave good biologic activity?Can the exogenous TRIM5a protein Antivirals against HIV-1 in vitro? These questions are very valuable for us,however,there is no report about that till to now.Matthew Stremlau's research of had shown that mutated TRIM5 a H(R328-332)[I→M(328),G→Q(330),R→P(332)]transfected by by retroviral vectors had a better function of Antivirals against HIV-1 than TRIM5α.Our laboratory has constructed the prokaryotic expression vector with PTD and conducted a preliminary study on the expression,purification and function of the protein.Objective:Cloning,sequence analysis and constructing the molecular phylogenetic tree of human TRIM5αgene of innate immunity.To construct a prokaryotic expression vector carrying PTD-TRIM5αand TRIM5αH(R328-332) gene,To express them in E.coli.and to explore the function of Antivirals against HIV-1 in vitro of new recombinated proteins.Methods:RT-PCR was employed to clone the human TRIM5αgene.Genomic structure was analyzed by comparing it with human genomic sequence.BioEdit,Genedoc and MEGA4.1 were used for alignment and phylogenetic analysis.PTD-TRIM5α/ pET28a and TRIM5αH(R328-332)/pET28a were constructed and were cloned into the expression vector pET 28a(+),transformed to E.coli BL21(DE3) strain,of PTD-TRIM5αand PTD- TRIM5αH(R328-332) proteins were induced and expressed,purified with Ni²⁺ chromatography.The expression and purification of PTD-TRIM5αand PTD-TRIM5αH(R328-332) were analyzed by SDS-PAGE and Western blot.Dilution and dislysis were used to renaturated the new recombinated proteinsThe interaction between PTD-TRIM5α/PTD-TRIM5αH(R328-332) and HIV-1Gag was detected by co-immunoprecipitation,His pull down and ELISA.Molt4 cell line was used to test the transmembrane capability of PTD-TRIM5α/PTD-TRIM5αH(R328-332) by fluorescence microscope.HIV-1_â…¢B mixed with target cell,then stained cells were treated with serially diluted drugs,anti-HIV-1 activity was oberserve by syncytial formation inhibition assay; viral duplication inhibition rateis was evaluated by measuring the levels of p24 antigen in culture supernatant by quantitative enzyme-linked immunosorbent assay(ELISA) kit.Result:A DNA fragment of 1482bp was cloned and sequencing analysis indicated that it carried an entire open reading frame and encoded a protein of 493 amino acid residues. The deduced amino acid sequence of human TRIM5αshowed similarities of 98.2%, 96.8%,93.7%,87.6%with those from Pan troglodytes,Gorilla gorilla,Pongo pygmaeus, Macaca mulatta,respectively.PTD-TRIM5/pET28a and PTD-TRIM5αH(R328-332)/pET28a were constructed and were cloned into the expression vector pET 28a (+).The expression and purification of PTD-TRIM5αand PTD-TRIM5αH(R328- 332) were confirmed by SDS-PAGE and Western blot.The new recombinated proteins in inclusion body was renaturated by dilution and dislysis.The study demonstrates that the human PTD-TRIM5αand PTD-TRIM5αH(R328-332) interacts with HIV-1 Gag in vitro.High transmembrane effectiveness of PTD-TRIM5αand PTD-TRIM5αH(R328-332) on Molt4 cells were verified by fluorescence microscope The date demonstrated that PTD-TRIM5αand PTD-TRIM5αH(R328-332) can inhibit the formation of syncytia,and the EC50 of PTD-TRIM5αand PTD-TRIM5αH(R328-332) against the formation of syncytia of Molt4 induced by HIV-1_â…¢B were 81.47μg/mL,7.55μg/mL resperctively.The EC50 of PTD-TRIM5αand PTD-TRIM5αH(R328-332) against p24 were 62.04μg/mL,4.74μg/mL resperctively.Conclusions: The human TRIM5αgene was cloned successfully.E.Coli.expression system of PTD-TRIM5 a and PTD-TRIM5 a H(R328-332) was constructed successfully and bioacitive PTD-TRIM5 a and PTD-TRIM5 a H(R328-332) proteins were obtained by expressoin,separation and purification.High transmembrane effectiveness of PTD-TRIM5αand PTD-TRIM5αH(R328-332) on Molt4 cells were verified by fluorescence microscope which offering a initial feasibility of the designed proposal,and the study demonstrates that TRIM5α/TRIM5αH(R328-332) interacts with HIV-1 Gag,PTD-TRIM5αH(R328-332) has more than10 times higher anti- HIV-1_â…¢B activities than TRIM5α.in vitro.The anti- HIV-1_â…¢B activities of the PTD-TRIM5αand PTD-TRIM5αH(R328-332) are connected with virus capsid(Gag) binding of the two proteins.