The Construction And Identification Of Eukaryotic Expression Vector And Fusion Protein Expression Vector Containing Human Folate Receptor Alpha Gene

Ovarian carcinoma accounts for more than half of the deaths due to gynecological malignancy with a 5-year survival rate of only 20-30%. The poor prognosis in ovarian cancer is generally due to the advanced stage of the disease at the time of diagnosis. After been treated with the combination of extensive cytoreductive surgery and platinum-based chemotherapy, the overall 5-year survival has remained below 40% during the last 3 decades. With the development of therapeusis, immunotherapy and targeting therapy have been in the focal point, and that has prompted the search for tumor specific antigen or tumor associated antigens which can be used as targets for MAbs for both diagnostic and therapeutic purpose.Human folate receptor 1 is a member of the folate receptor (FOLR) family. There is clear evidence that FOLR-1 expressed at very low levels on the cell surface of nomal epithelial tissues. Overexpression of FOLR-1 has been detected on -90% of ovarian carcinomas; in 20-50% of breast, colorectal, lung, and in multiple other tumor types, the level of expression is usually >20-fold normal tissue expression and has been reported to be as high as 80~90-fold in ovarian carcinomas. In thefollowing works, researchers found the immunodominant E39 and subdominant E41 epitopes are efficient at amplifying the response of tumor-associated lymphocyte populations in terms of lytic function, enhanced proliferation, and specific IFN-gamma release, suggests the exciting potential of a widely applicable FBP-based vaccine in epithelial cancers.In the first part of this research, we construct the clone vector of FOLR-1 and the polynucleotide vaccine based on the gene of FOLR-1. FOLR-1 gene was amplified from human ovarian carcinoma cell SKOV3 by RT-PCR, the product of PCR was ligated into pGEM-T vector after restriction endonuclease digestion. Gene fragment encoding FOLR-1 was correctly inserted into the vector, which was determined by an automated DNA sequencer and restriction endonuclease digestion, and then was subcloned to corresponding sites cut with EcoR I plus Xho I of eukaryotic expression vector pcDNA3.1(+). The vector pcDNA-FRl was transfectde into HO-8910 cell and been detected by RT-PCR in transcription level.In the second part, the gene of FOLR-1 was inserted into a fusion protein expression vector pGEX-4T-2, in which the foreign gene was controlled by Ptac promoter. The recombinant plasmid pGEX-FRl was transferred into E.coli DH5 a and expression was induced with IPTG. After induction, bands of Mr = 55,000 appeared on 10% SDS-PAGE gel and Western-blot filter paper. And the expression level is 11.5%.In this study, we constructed the recombinant FOLR-1 eukaryotic expression vector (pGEX-FRl) and fusion expression vector (pcDNA-FRl). The successful construction and expression of pcDNA-FRl and pGEXFRl provide an experimental basis for earlier diagnosis, targeting thearpy and immunotherapy for ovarian carcinoma.