重组人B7-H1IgV肿瘤疫苗的发酵纯化工艺研究和抑瘤活性的检测

发布时间：2018-05-24 03:21

本文选题：重组 + B7-H1　；参考：《第四军医大学》2010年硕士论文

【摘要】： 目的:B7-H1是共刺激分子B7-H1家族成员,B7-H1主要通过其IgV样区与受体PD-1结合发挥免疫抑制作用,参与肿瘤的免疫逃逸。近年来的研究表明,阻断B7-H1,可以显著的抑制肿瘤的生长。本实验室先前构建了重组人B7-H1IgV蛋白,检测免疫该蛋白小鼠的抗血清活性及蛋白疫苗对荷瘤小鼠肿瘤生长的抑制作用,表明重组人B7-H1IgV蛋白疫苗既可以诱导机体产生特异性的抗B7-H1抗体和肿瘤特异性的细胞免疫,又能协同其它肿瘤疫苗发挥的抗肿瘤作用。为了进一步加快该制品的产业化生产,本课题拟通过全面检定工程菌,建立稳定的重组人B7-H1IgV工程菌菌种库,并建立稳定的发酵纯化生产工艺。方法:首先,通过对重组人B7-H1IgV工程菌进行重组质粒酶切鉴定和序列分析、IPTG诱导后表达水平的检测、连续传代50代的工程菌每隔10代进行携带质粒及其表达目的蛋白稳定性的检测,形态学的电镜观察,革兰氏染色检定和生化反应的检定,建立稳定可靠的工程菌菌库;然后依次通过5L发酵罐连续三批工程菌的发酵,裂菌,包涵体溶解,建立稳定的发酵工艺,再通过目的蛋白的复性,两次Ni亲和层析纯化,建立稳定可靠的纯化工艺;最后通过动物实验对生产的目的蛋白的免疫原性,抑瘤活性进行检测。结果:工程菌重组质粒的酶切和测序结果与预期一致,目的蛋白表达量达到10%以上,工程菌连续传50代,每隔10代的工程菌质粒酶切结果和目的蛋白表达水平均一致,电镜结果显示工程菌呈明显的大肠杆菌形态,且无其它微生物的污染,革兰氏染色结果显示工程菌呈革兰氏阴性,各项生化反应检定结果表明工程菌为大肠杆菌。5L发酵罐连续发酵3批工程菌均可得到约190克湿菌,且各批菌表达目的蛋白水平均在10%以上。添加GSH/GSSG氧化还原系统参与蛋白复性,显著提高了复性过程目的蛋白的可溶性,两次Ni亲和层析纯化目的蛋白,纯度达到95%以上。B7-H1IgV蛋白免疫小鼠,两周内抗B7-H1的抗血清可达到1×104以上,B7-H1IgV能够显著抑制荷瘤小鼠的肿瘤生长,抑瘤率达到30%。结论:重组人B7-H1IgV工程菌稳定可靠,发酵和纯化生产工艺简单,稳定,经济,生产制备的蛋白疫苗抑瘤活性强。本研究结果为重组人B7-H1IgV蛋白的大规模生产和系统的临床试验奠定了基础。
[Abstract]:Objective: B7-H1, a member of the B7-H1 family of costimulatory molecules, plays an immunosuppressive role by binding its IgV like region to the receptor PD-1, and participates in the immune escape of the tumor. Recent studies have shown that blocking B _ 7-H _ 1 can significantly inhibit tumor growth. The recombinant human B7-H1IgV protein was previously constructed in our laboratory to detect the antiserum activity of immunized mice and the inhibitory effect of protein vaccine on tumor growth in tumor-bearing mice. The results showed that the recombinant human B7-H1IgV protein vaccine could not only induce specific anti-B7-H1 antibody and tumor-specific cellular immunity, but also cooperate with other tumor vaccines. In order to speed up the industrial production of the product, this paper intends to establish a stable recombinant human B7-H1IgV engineering strain bank and establish a stable fermentation and purification production process through the comprehensive identification of engineering bacteria. Methods: firstly, the recombinant plasmid was digested and sequenced to detect the expression level of recombinant human B7-H1IgV. A stable and reliable engineering bacterial library was established by detecting the stability of the plasmid carrying plasmid and its expressed target protein, observing the morphology by electron microscope, detecting the Gram staining and biochemical reaction of the engineering bacteria from 50 successive passages every 10 generations. Then, three batches of engineering bacteria were fermented in 5L fermenter in succession, the bacteria and inclusion bodies were dissolved, a stable fermentation process was established, and then the stable and reliable purification process was established by renaturation of the target protein and purification by two Ni affinity chromatography. Finally, the immunogenicity and antitumor activity of the target protein were detected by animal experiments. Results: the results of enzyme digestion and sequencing of recombinant plasmids of engineering bacteria were consistent with expectations. The expression of target protein was more than 10%, and the expression level of target protein was the same as that of plasmids digested every 10 generations. The results of electron microscope showed that the engineering bacteria showed obvious form of Escherichia coli and no contamination by other microbes. The results of Gram staining showed that the engineering bacteria were gram-negative. The results of various biochemical reactions showed that the engineering bacteria were E. coli. 5L fermenting continuously for 3 batches, about 190 grams of wet bacteria could be obtained, and the expression level of target protein of each batch of bacteria was more than 10%. Adding GSH/GSSG redox system to the protein renaturation significantly increased the solubility of the target protein. The purified protein was purified by twice Ni affinity chromatography, and the purity of the protein was over 95%. B7-H1 IgV protein was immunized in mice. Within two weeks, the antiserum against B7-H1 could reach more than 1 脳 10 ~ 4 B7-H1 IgV, which could significantly inhibit the tumor growth of tumor-bearing mice, and the tumor inhibition rate was 30%. Conclusion: the recombinant human B7-H1IgV engineering strain is stable and reliable, the production process of fermentation and purification is simple, stable and economical, and the protein vaccine has strong anti-tumor activity. The results laid a foundation for mass production of recombinant human B7-H1IgV protein and systematic clinical trial.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392;Q819

【相似文献】