Cloning And Construction Of Mammary Gland Expression Vector For Human Serum Albumin

Human serum albumin (HSA) is the most abundant protein in huaman bloodplasma. HSA takes an important role in human bodies.The new method of producing gene-medicine is through making mammary gland bioreactor producing medical protein with the safe , effective and low cost prospect starting point .That is to say it is promising .In this research,the mammary gland-specific expression vector of human serum albumin was constrcted ,which was partial consisted of the conducting elements including the 5′and 3′regulation region of bovine ?-casein,the expression sequence of HSA cDNA, antibiotics gene (Kana/Neo) and report gene (EGFP).so it can be used to trans-fect mammary epithelial cells. Positive clone cells were selected and purified, So the cells can be used as sources to create transgenic animals by nuclear transplant techniques. The results of research is following: 1.According to the cDNA sequence of HSA(human serum albumin HSA) derived from the Genebank(v00495), a pair of primers was designed. RT-PCR applying to amplify the whole 1 995 bp cDNA of HSA gene.The DNA fragment was cloned into vector pMD-18T and transformed into E.coli DH5a host bacteria. XbaⅠendonuclease digestion and sequencing from selected positive cloned bacteria. The fragment was sequenced, and it was compared with serum albumin cDNA gene of human, monkey, cow,swine, sheep and mouse registered in GenBank . The results indicated the homology were 99 %, 92 %, 82 %, 82 %, 81 % and 75 % respectively. The mutation doesn,t change the most open reading frame,that is to say the HSA cDNA is cloned successfully. 2 . The total RNA was harvested from human liver tissue with Trizol. The 28srRNA ,18srRNA and5 srRNA strips were identified with the help of formaldehyde gelatin denaturalization electrophoresis.It indicated the RNA integrality is proper and the RNA can be used in RT-PCR. 3.A new pair of primer was designed according to multiple cloning sites of pEGFP-C1 vector and the restriction endonuclease site of the beta-casein promoter sequence. The HSAcDNA was added restriction endonuclease NheⅠand BamHⅠsite. Using the strategy of directional clone after double digestion by different endonucleases,the requisite expressing sequence of human cDNA was merged with the beta-casein promoter sequence. The fusion vector was sequenced, and the result showed that there were no mutants in expressing sequence of human serum albumin cDNA, which also had a proper reading frame. 4 . Using the strategy of directional lone after double digestion by different endonucleases,while utilizing a eukaryotic vector pEGFP-C1, the non-fusion type of mammary gland expression vector pEGFP-BL for human serum albumin was constructed, which also included antibiotics gene (kana/neo) and report gene (EGFP). The plasmid pEGFP-BH was verified by PCR and endonuclease digestion. And the result showed that the insertion place of serum albumin expressing sequence in the vector is just right. 5.The plasmid pEGFP-BH was transfected into mammary epithelial cells cultured in vitro using liposome. After preliminary screening by G418, the expression of report gene EGFP mainly in cytoplasm was observed under fluoroscope. A pair of detecting primers was designed, and the positive cloned cells were studied through PCR. The result indicated that the whole expression plasmid possibly had been integrated into the chromosome of positive mammary epithelial cells.