| There are a lot of market demanding at home and abroad about Human serum albumin (HSA).HSA takes an important role in human bodies.The new method of producing gene-medicine is through making bird bioreactor producing medical protein with the safe ,effective and low cost prospect starting point . That is to say it is promising.It is important for bioreactor studying of birds is to built the specific expressional vector which can control the expressing of exogenous gene.The establishment of upstream vector is the first important element. The expression of exogenous gene in oviduct of quail must have the participating of tissue-special promoter. In this research, using 5'-flanking of ovalbumin in quail for the control sequence to activate the expression of HAS,. Antibiotics gene (Kana/Neo) and report gene (EGFP). So it can be used as sources to create transgenic animals by nuclear transplant techniques. The results of research is following:1 The cloning of Human serum albumin.About 1.8kb sequence of the fragment of human serum albumin containing were successfully amplified by RT-PCR..the purpose of the initia gene were identified with the help of electrophoresis. The DNA fragment was cloned into vector pGEM-T and transformed into E.coli DH5a host bacteria. Xbaâ… e ndonuclease digestion and sequencingfrom selected positive cloned bacteria. The fragment was sequenced, and it was compared with serum albumin cDNA gene of humanin GenBank.The results indicated the homology were 97 %,The mutation doesn,t change the most open reading frame,that is to say the HSA cDNA is cloned successfully.2 Construction of quail oviduct-specific expression vector of human serum albumin gene. (1)Using 5'-flanking of ovalbumin in quail for the control sequence to activate the expression of HAS. A new pair of primer was designed. TheHSAcDNA was added restriction endonuclease Kpnâ… and Hindâ…¢site. Using thestrategy of directional clone after double digestion by different endonucleases,the requisite expressing sequence of human cDNA was merged with the 5'-flanking of ovalbumin in quail. With restrictive interior contact enzyme kpnâ… With Hindâ…¢cuts material particle pOVand pHSA, then connects reorganization material particle for pOV-pHSA with the T4 ligase. The fusion vector was sequenced by enzyme digestion.(2)Using the strategy of directional lone after double digestion by different endonucleases, while utilizing a eukaryotic vectorpIRES2-EGFP, the non-fusion type of oviduct expression vector pEGFP-VH for human serum albumin was constructed,which also included antibiotics gene (kana/neo) and report gene (EGFP).The plasmid pEGFP-VH 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.3 The expression of Human serum albumin gene in in primary oviduct epithelium and embryo fibroblasts cells(QEF)of quail.(1)we constructed the primary oviduct epithelium and quail embryo fibroblasts cell ( QEF ) of quail were cultured as testing platform. Post-recombinant expression vector of human serum albumin transfected into the two cells using liposome. The expression of report gene EGFP mainly in cytoplasm was observed under fluoroscope. The results showed thatthere was no expression at fibroblasts of the recombination vectors, but it was expressed in oviduct epithelium primary.(2) Genomic DNA of transfected cells were extracted,specific primers were detected,reporter gene-positive cell clones were detect by using PCR method,and molecular weight of Quail oviduct epithelial cell secretory protein were fixed by using SDS-PAGE.The results showed that there were HAS which molecular weight is 68kDa in transfected quail oviduct epithelial cellculture supernatant,which proved that the constructed expression vector was effective.And the expression of the initial components have been integrated into the quail oviduct epithelial cells positive for the chromosome.
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