The Study On Construction Of Chicken Zyxin Slow Virus Infection And Gene Eukaryotic Expression Vector And Chicken Caecum Tissue Culture

Zyxin (zyxin), also known as zyxin2, is an intracellular protein. During mitosis, zyxin gene and other genes form a way of regulating complex to control process of mitosis. In addition, zyxin is also as an activator of transcription in the nucleus for transcription work. Zyxin synergies with other proteins, regulate mitosis and cell movement, migration and growth of tumor cells. Although zyxin gene in chickens and humans only are about60%of gene sequence homology, they have a high functional similarity. According to the QTL and polymorphism analysis has demonstrated that the gene is associated with Coccidiosis-resistance. This research to build zyxin gene eukaryotic expression vector and slow virus infection carrier and filter its effective jamming target, while exploring the way of tissue culture conditions of chicken cecum organization, to lay the foundation for the future verification of the Coccidiosis-resistance and mechanism study on the Coccidia infection.Using trizol RNA extraction thymus tissue in jinghai yellow chicken,by the way of RT, using RT Kit to generate cDNA.set cDNA as template,use the PCR primer containing NheI and SalI restriction endonuclease sites and protect base to PCR and gel recycling to get zyxin gene fragments. Cut zyxin gene fragment and Pex-6using NheI and SalI,and gel recycling to get the product. use T4DNA ligase enzyme products to connect. connect products transform competent Escherichia coli, take positive clones sequenced. Extracte the plasmid vectors which are sequenced to be correct to get Pex-6-zyxin vectors. Transfect Eukaryotic expression vector Pex-6-zyxin into293T cells and determine its expression level by Real time-PCR. These indicate that:1. the design of PCR amplification primers annealing temperature of55degrees Celsius successfully expanded to zyxin gene. Electrophoresis results indicate that increased with the purpose of (1629bp) is located between the DNA molecular weight Marker1500-2000, in line with the expected size;2. enzyme cut zyxin gene fragment and pEX-6carrier respectively with Nhel and Sall,then connect two enzyme cut product by T4connection enzyme. Transfer it into Escherichia coli.Extract plasmid, after double enzyme cut identification, purpose gene stripe (with size for1629bp) and linear carrier stripe (with size for4.88kb) are located in DNA Marker1500-2000and4000-5000respectively,which prove that restructuring plasmid is Pex-6-zyxin. Successfully built Pex-6-zyxin eukaryotic expression vector.3. After the transfection of recombinant vector into293T cells, we can see visible red light under fluorescence microscope.it demonstrates that Pex-6-zyxin is successfully transfected into293T cells and transfection efficiency is higher than80%. The48hours and72hours after recombinant plasmid transfected into293T cells, determine then by Real time-PCR respectively.after testing, it turns out zyxin gene has expression in the293T cells. histogram display,48hours zyxin gene expression between400and450,72hours zyxin gene expression is500,which reveals that zyxin gene expression level is on the rise among48hours to72hours.Design fragment4interference fragments by Oligo Designer3.0, synthesis them by the Shanghai Jima company. Annealing forward and reverse templates to get shRNA. Double digestion Lv3and shRNA,Using BamHI and EcoRI and connect the inerference fragments and Lv3vector by T4NDA ligase enzyme. cotransfect Lv3-shRNA vectors and packaging plasmids which include pGag/Pol、pRev and Pvsv-G into293T cells to package virus.Dilute virus liquid10times the6gradients, and transfect them into293T cells respectively to determine the titer. Transfect4recombinant lenti-virus interference vectors and eukaryotic expression vectors Pex-6into293T cells, and filter the effective interference target by RT-RCR and Western-blot. These indicate that:1.,After BamHI and EcoRI double enzyme Lv3carrier, the electrophoresis results indicate size of8kb, in line with expectations. Building4zyxin lentivirus shuttle plasmid expression vectors CO1, CO2, CO3, CO4and one negative control plasmid for zyxin gene. EcoRI cuting and sequence identification show it is correct, successfully built zyxin Lv3-shRNA recombinant plasmid; 2. recombinant Lentiviral vector plasmid Lv3-shRNA and Pack mixture of plasmid transfected293T cells together and observe cell under inverted fluorescence microscope.After24hours,it shows fluorescent green,cell growth is good,which indicates that virus package successfully.93T cells transfected with total packaging and concentrated the titer of the virus1×108TU/ml,which shows it is suit to infect293T cells. CO3interference efficiency is60%at RNA level and90%at the protein level. comparing other RNAi lentivirus shuttle plasmid expression vectors, CO3interference works best. Successful build zyxin gene Lentiviral expression vector of RNA interference, and screen the effective interference targets, laid the Foundation for further study of zyxin gene function.Disinfect18days fertilization eggs of jinghai yellow chicken by75%alcohol.Get out chicken cecum,cut them into paste, transfer them into microcentrifuge tube in Sterile console.put DMEM which contains penicillin and streptomycin into tube, centrifugal, discard the supernatant liquid. Culture Organization blocks using DMEM with3%FBS. Extract mRNA,RT,determine expression level of zyxin gene at36hours and72hours respectively. These indicate that:1. in the design of this study under the culture medium components, tissue growth is good in the cultivation of the8th day, tissue and cells covered80%of the flat dish, although the constant stream of cells died off, but there will be new cells growth, apoptosis of cells gradually in the future.Cells die gradually after8days.2.zyxin express in the36hours and72hours cecum tissue cell cultures,and72hours expression level is significantly lower than that of36hours.