Construction Of The IRES-based Screening Vector For Multiple Gene Co-Stable Expression And MDCK Cell Lines Stably Expressing Siat7e Gene

In scientific research,construction of stable expressing cell lines,and screening seed cells in fields of antibody engineering and cell engineering,often need two or more genes to co-stable express.Co-transfection of two vectors into cells can achieve gene expression,but this strategy will face many problems.Therefore,it is necessary to develop a single vector to achieve multiple genes co-expressing.MDCK cell is one of the important cell lines for the production of influenza virus vaccines,it is widely used in amplification and purification of a variety of virus.But MDCK cell is anchorage-dependent and thus requires surface adhesion to proliferate.The achievement of MDCK cells growing in suspension will simplify the production process and amplify the scale of production of influenza virus.The first part of this article is construction of the IRES-based screening vector for double gene co-stable expression.Objective: An IRES-based vector was constructed to achieve co-expression of two target genes with the screening marker gene promoted by the single promoter,and to improve the screening efficiency of multiple genes co-stable expression cell lines.Methods: A bicistronic expression element BamHI-MCS1-IRES-MCS2-IRES-BsiWI which has two multiple cloning sites was designed and synthesized.The vector named pLV-2MCS-Puro was constructed by inserting the element into the skeleton vector pLV-MCS-Puro which was constructed previously in our lab.The DsRed2 and EGFP genes were inserted simultaneously into the vector to test the screening efficiency of multiple genes co-stable expression cell lines.Results: The vector pLV-2MCS-Puro and the recombinant plasmid pLV-DsRed2-EGFP-Puro were constructed successfully.Transient transfection experiment showed that the vector can mediate co-expression of multiple genes.MDCK and Hela cell pools resistant to puromycin were obtained through transfection of the recombinant plasmid.The fluorescent inverted microscope showed that DsRed2 gene at the upstream of the IRES sequence and EGFP gene at the downstream of IRES sequence wereco-expressed in cells,and the double positive rate was close to 100%.It indicated that this vector has high screening efficiency.The results of genomic PCR,RT-PCR and Western Blot showed that DsRed2 and EGFP genes were stably integrated into cell genome and the two proteins were expressed consistently.Conclusion: The IRES-based vector pLV-2MCS-Puro was successfully constructed and proved to be efficiently in screening multiple genes co-stable expression cell lines.This vector will have certain application prospects in studying protein interactions and constructing engineering cell lines.The second part of this article is construction of MDCK cell line that stably expresses of siat7 e gene.Objective: In this chapter,MDCK cells expressing siat7 e gene was constructed to solve the question of suspension culture.Methods: The full-length siat7 e gene of 1011 bp was amplified from the MDCK cells genome by PCR,and the gene was cloned into pBflag,pLV-MCS-Puro and pSF-EGFP vectors,respectively.Three kinds of eukaryotic expression vectors were transfected into MDCK cells.MDCK cells that stably expressed siat7 e gene were screened,in order to achieve MDCK cells adapting to suspension culture.Results: Three kinds of eukaryotic expression vectors pBflag-siat7 e,pLV-siat7e-Puro and pSF-EGFP-siat7 e were successfully constructed.Four MDCK clones resistant to G418 were obtained through transfection of the recombinant plasmid pBflag-siat7 e.Results of western blotting and genomic PCR didn't show siat7 e target bands,suggesting that siat7e-positive clones may not be screened,and no obvious phenotype was presented.MDCK cell pool resistants to puromycin were obtained through transfection of the recombinant plasmid pLV-siat7e-Puro.Results of genomic and transcriptional levels of PCR demonstrated that the target gene siat7 e was stably integrated into the MDCK cell genome.No obvious phenotype was presented similarly.Drug-resistant MDCK cells were cultured by low serum or serum-free medium.Unfortunately,MDCK cells adapted to suspension culture were not obtained.Recombinant vector pSF-EGFP-siat7 e was constructed,integrating EGFP and siat7 e protein.The siat7 e protein was screened byfluorescence.The fluorescent inverted microscope showed that EGFP gene was expressed in cells.It indicated that siat7 e gene was successfully transfected into MDCK cells.Similarly,results of genomic and transcriptional levels of PCR demonstrated that the target gene siat7 e was stably integrated into the MDCK cell genome.Fluorescence co-localization assay confirmed that the protein expressed by siat7 e gene was located on the endoplasmic reticulum.Conclusion: Three kinds of vectors were successfully constructed,and two kind of drug-resistant MDCK cells that stably expressing siat7 e gene were obtained.Localization of sialyltransferase was successfully verified,but no obvious phenotype was presented.Next,the characteristic that MDCK cells adapt to suspension culture will be further studied.The achievement of MDCK cells growing in suspension will will be beneficial for large-scale production of influenza vaccines.