The Construction Of Mammary Gland Specific Expression Vector Of E2 Gene Of Classical Swine Fever Virus And Expression Study On Cell

Classical swine fever is a harmful disease to swine husbandry.Classical swine fever virus (CSFV) is its cause.And its main protective antigen is glycoprotein E2.Animals can prevent from deadly virus's attack if they vaccinate protein E2 coming from eukaryotic expression system aheadly.Producing bioactive proteins through mammary gland bioreactor is a hot focus in the field of biology,and the key question is the construction of expression vector in mammary gland.Beta-lactoglobulin (BLG) is the main protein in ruminant milk. For the purpose of constructing mammary gland specific expressional vector,we use the updream regulation sequence of bovine BLG gene as promoter to regulate CSFV E2 gene to express in mammary epithelial cells in vitro in order to verify the expression efficiency of foreign gene and rationality of expressional vector.Positive clone cells are selected and purified by the expression of antibiotics gene Kana/Neo and report gene EGFP.So the cells can be used as sources to create transgenic animals by nuclear transfer techniques.The results of research are as following:Bovine BLG gene updream regulation sequence is cloned by method of PCR from cow blood.It consists of 5' flank sequence about 645bp and the signal peptide sequence in first exon about 92bp.After PCR product is recovered and purified,it is cloned into T site of pMD 18-T Vector.Then it is sequenced,and compared with other bovine BLG gene in Genbank.The results indicate the homology are all above 90.00%.At last the sequence is analyzed by computer,we find that many factors or protein binding sites and response element,such as mammary specific binding factor (MSBF),nuclear factor-1 (NF-1),retinoic acid response element (RARE) and TPA response element (TRE)are include in it.It suggestes the cloned regulatory element could direct foreign genes to express specifically in mammary gland epithelia cells,and it could be used to construct mammary gland-specific expressional vector.BLG regulation sequence and CSFV E2 gene are linked by method of recombinant PCR,and then they are inserted into pMD 18-T vector.The positive recombinant plasmid is marked as pBE.The composed fragment BE is obtained by digesting pBE with restrictive enzyme HindⅢ/XbaⅠ and AseⅠ.Vector pEGFP-C1 is also digested by AseⅠ,and is linked with BE fragment by T4 DNA ligase after its 5′end is dephosphatased.So the mammary gland specific expressional vector is constructed,which contains report gene (EGFP),antibiotics gene (Kana/neo),regulatory element (bovine BLG gene upstream regulation fragment) and foreign target gene (CSFV E2 gene). The recombinant plasmid are transfected into bovine mammary epithelial cells cultured in vitro by electrotransfection.Positive clone cells are selected and purified by G418.The expression of report gene (EGFP) are observed under fluoroscope.The results show the expressional vectors containing CSFV E2 gene are successfully transfected into bovine mammary epithelial cells,and the report gene express according to presupposition.