The Development Of Rhb7-h1igv Tumor Vaccine

Objective: B7-H1, a member of B7 family of co-stimulatory molecules, mediate immunosuppression by binding its receptor with B7-H1IgV domain, participating in tumor immune escape. Blockaed of B7-H1, could delay tumor growth and prolong the survival on tumor-bearing mice. Our previous study constructed B7-H1IgV vaccine, which could induce high titer of anti-B7-H1 antibody, inhibit tumor growth. To further speed up the industrial production of the product, the aim of this study is establishing rhB7-H1IgV is to establish a stable recombinant human B7-H1IgV engineering bacteria strain library by comprehensive test, and tne stable production technology of fermentation and purification. Methods: First, the recombinant human B7-H1IgV plasmid was tesed by restriction enzyme digestion and sequence analysis. The recombinant strain was subcultured for 50 passages, and the expression level of B7-H1IgV, property of plasmid,as well as morphology,cultivation characters and biochemical reactions of various passages were studied. Then developed a procedure,consisting of high density fed-batch culture by consecutive three batches of fermentations in 5L fermenter, isolation of inclusion bodies, gradient refolding, followed by Ni affinity chromatograph for twice. At last, detect antitumor activity of B7-H1IgV vaccine by animal experimrnts. Results: The restriction enzyme digestion and sequencing results of pQE30-rhB7-H1IgV consisitented with expectations. The protein expression level reached 10%. pQE30-B7-H1IgV/DH5αshowed a typical morphology of E.coli, and the characteristics of various passages were not significantly different from that of primary strain; Consecutive three batches of fermentations in 5L fermenter harvested about 190g bacteiral respectively and the protein expression level reached 10%. GSH/GSSG redox system significantly improved protein solubility in renaturation The purity of the purified rhB7-H1IgV by HPLC was over 95%. The result of Western blot was identical with thoses required. Animal experiment indicated that, rhB7-H1 not only induced high titer of anti-B7-H1 mAb which was over 1×104,but also inhibit tumor growth. The tumor inhibition rate exceeded 30%. Conclusion: The strain is suitable for large-scale production. The whole pilot procedure was simple and fast, and the equipment needed was cheap, so it was promising to enlarge development.