The Screening And Identification Of CD4Interactive Proteins In Mice (Mus Musculus)

Research Background:Mammal sperm could interact and transfer the exogenous DNA, Sperm-mediated gene transfection （SMGT） is a convenient, effective method to produce transgenic animals. However, this method has a great of random and uncertain characters. The main problem that restricts it to be extensively used and promoted is the unclear mechanism of sperm interacting and transferring the exogenous DNA. Currently, there are not many researches on it and most of them concentrate on CD4, a molecule which is on the membrane of sperm. CD4is one of the receptor and transfer molecule on sperm membrane. It is a single chain cell membranesurface protein. Professor Lavitrano, the founder of SMGT had confirmed that CD4molecule had an important function in the process of sperm interacting and transferring the exogenous DNA, as well as our previous studies. Whereas, the sperm which closed CD4molecule still partly had the fuction to transfer exogenous DNA. This demonstrated that there not only CD4molecule, but also other molecules to help exogenous DNA to transfer to sperm, CD4and them interacted with each other to finish this process. Proteins interactions have a key role in life activities, therefore, screening the molecules which interact with CD4and have an important role in interacting and transfering exogenous DNA will support a new direction to reveal the mechanism of sperm-mediated gene transfer and establish a stable, simple, efficient sperm mediated transgenic technology foundation.Materials and Methods:At first, we constructed bait plasmid vector pGB-mCD4. The concret methods included extracting mRNA from mouse testis; mCD4gene was acquired by using RT-PCR protocol; mCD4connected with the plasmid vectors pGB then the positive clone plasmid vectors sequenced by Shanghai Sangon, and they were performed the positive self-activity detection. Then, we used yeast two hybrid system to screen the Mouse Testis cDNA Library. The positive clones were sequenced and compared by GenBank. Screening genes which had interactions with CD4had been performed primarily bioinfrmatics analysis. Lastly, we tested the co-expression of mCD4and4kinds of candidate molecules in mammal293FT cells. The concret methods included constructing plasmid vecors pCDEF-Myc-mCD4, pCDEF-FLAG- BSPRY, pCDEF-FLAG-RANBP9, pCDEF-FLAG-CD320, pCDEF-FLAG-Creld2, then, we transferred the constructing vectors into the293FT cells, transfected48h, we used fluorescence microscope and FACS to determine the transfecton efficiency. At last, we verificated the cell lysate by using western blot.Results:（1） Constructing plasmid vector pGB-mCD4, the length of mCD4was123bp, genes were electrophoresed, sequenced and then they were confirmed as target fragments; bait vector was transferred into Y190strains, after β-galactosidase developing reaction, we found that pGB-mCD4could not activate the yeast cells Y190.（2） The transfection efficiency of testis cDNA library was9.3×105cfu/uμg. Culturing the transfected yeast cells Y190, there were199clones could grow on the Leu-Trp-His-/SD+3AT medium plates, subsequently, the clones were analysed by β-galactosidase developing reaction. The results showed that there were26positive clones as their colours changed blue through the developing reaction. We transferred them into bacteria competence and cultured them on the LB+Ampr plates, then, we chose the clones to amplify the bacteria plasmid. Every positive clone was co-trasfered with bait vector pGB-mCD4into yeast cells Y190. After that, the positive clones were analysed by β-galactosidase developing reaction and23simples changed blue. Every gene was sequenced and compared by GenBank. At last, we got8positive genes, they were BSPRY （Accession Numbers: NM₁38653.1）, Pmpcb （Accession Numbers:NM₀28431.2）, RANBP9（Accession Numbers: NM₀19930.2）, Morc2b （Accession Numb-ers:NM₁77719.4）, Ggnbp2（Accession Numbers:NM₁53144.2）, CD320（Accession Numbers:NM₀19421.3）, Adam2（Accession Numbers: NM₀09618.2） and Creld2（A-ccession Numbers: NM₀29720.2）.（3） We constructed plasmid vectors including pCDEF-Myc-mCD4, pCDEF-FLAG-BSPRY, pCDEF-FLAG-RANBP9,pCDEF-FLAG-CD320and pCDEF-FLAG-Creld2, they were co-transferred into the293FT cells, the transfecton efficiency were97.97%. The genes were verificated by Western blot, the result showed that BSPRY gene and RANBP9gene could express stably in293FT cells.Conclusions:Using yeast two hybrid system, total of8candidate molecules which interacting with CD4molecule were screened.