重组人白细胞介素-15的原核表达和治疗肿瘤的研究
发布时间：2019-01-01 16:56
【摘要】：白细胞介素-15(IL-15)是Grabstein等人于1994年发现的一种具有免疫调节活性的细胞因子，它具有与白细胞介素-2(IL-2)相似的生物学功能，可促进T细胞、B细胞、自然杀伤细胞(NK)的增殖与分化，增强细胞毒T细胞(CTL)／NK细胞的细胞毒活性等，并能发挥优于IL-2的抗肿瘤作用。 本论文是在我们的前期工作基础上，通过基因工程技术和蛋白质纯化技术获得了纯度在95％以上的重组人白细胞介素-15(rhIL-15)，并用建立的小鼠肿瘤模型，研究了rhIL-15的抗肿瘤作用，为将rhIL-15开发成一类抗肿瘤新药奠定了基础。本论文工作主要研究内容包括三部分：(1)rhIL-15的原核表达、纯化和鉴定；(2)抗rhIL-15单克隆抗体的制备；(3)rhIL-15的急性毒性实验和抗肿瘤作用研究。 (1)rhIL-15的原核表达、纯化和鉴定 根据已知人白细胞介素-15(hIL-15)成熟蛋白的氨基酸序列，，在不改变氨基酸序列的前提下，采用大肠杆菌偏爱密码子合成寡核苷酸片段，通过拼接PCR法成功合成hIL-15基因，分别插入原核表达载体pGEX-2T、pET_(30a)、pET_(42)、pETBlue-1或pBV_(220)，在多种宿主菌中获得了表达，最后筛选到稳定高效表达rhIL-15的工程菌株，即BL_(21)Star~(TM)(DE3)plysS／pBV_(220)-IL-15。在工程菌BL_(21) Star~(TM)(DE3)plysS／pBV_(220)-IL-15中，rhIL-15以包涵体形式表达，最高表达量为菌体总蛋白的15.2％。经蛋白印迹法鉴定表明，此rhIL-15能与抗hIL-15的单克隆抗体特异结合。诱导表达后，分离出包涵体，经反复洗涤后，溶于6mol／L盐酸胍，获得rhIL-15粗品。再经反相层析分离后溶于8mol／L尿素，经透析或稀释复性获得有生物活性的rhIL-15，复性后的rhIL-15依次经DEAE弱阴离子交换层析和Sephadex G-25脱盐得到纯化的rhIL-15。纯化的rhIL-15行SDS-PAGE鉴定及光密度扫描分析，结果显示，纯度在95％以上。N端氨基酸序列分析结果表明，此rhIL-15 N端的前15个氨基酸序列与预期的一致；毛细管电泳分析表明，纯化rhIL-15的纯度接近100％，等电点为4.14，接近rhIL-15的等电点理论值4.42。纯化rhIL-15的体外促CTLL-2细胞增殖活性为6.7×10~6U／mg。因此，可以确定分离纯化的表达蛋白即是rhIL-15。
[Abstract]:Interleukin-15 (IL-15) is a cytokine with immunomodulatory activity discovered by Grabstein et al in 1994. It has biological functions similar to that of interleukin-2 (IL-2) and can promote T cells and B cells. The proliferation and differentiation of natural killer cell (NK), the enhancement of cytotoxic activity of cytotoxic T cell (CTL) / NK cells, etc. On the basis of our previous work, recombinant human interleukin-15 (rhIL-15) with purity of more than 95% was obtained by genetic engineering and protein purification techniques, and the mouse tumor model was established. The anti-tumor effect of rhIL-15 was studied, which laid a foundation for the development of rhIL-15 as a new anti-tumor drug. The main contents of this thesis are as follows: (1) prokaryotic expression, purification and identification of rhIL-15; (2) preparation of monoclonal antibody against rhIL-15; (3) acute toxicity test and antitumor effect of rhIL-15. (1) prokaryotic expression of rhIL-15, purification and identification of amino acid sequence of human interleukin-15 (hIL-15) mature protein, without changing amino acid sequence, Oligonucleotide fragments were synthesized by Escherichia coli codon preference, and hIL-15 gene was successfully synthesized by splicing PCR, and inserted into prokaryotic expression vectors pGEX-2T,pET_ (30a), pET_ (42), pETBlue-1 or pBV_ (220), respectively. It was expressed in a variety of host bacteria. Finally, the engineering strain BL_ (21) Star~ (TM) (DE3) plysS/pBV_ (220)-IL-15. was screened to express rhIL-15 stably and efficiently. In the engineering strain BL_ (21) Star~ (TM) (DE3) plysS/pBV_ (220)-IL-15, rhIL-15 was expressed as inclusion body, and the highest expression amount was 15.2g of the total cell protein. Western blot analysis showed that the rhIL-15 could specifically bind to monoclonal antibody against hIL-15. After induced expression, the inclusion bodies were isolated, washed repeatedly and dissolved in 6mol/L guanidine hydrochloride to obtain rhIL-15 crude products. After being separated by reverse phase chromatography and dissolved in 8mol/L urea, the rhIL-15 with bioactive rhIL-15, was obtained by dialysis or dilution renaturation. The purified rhIL-15. was obtained by DEAE weak anion exchange chromatography and Sephadex G-25 desalination. The purified rhIL-15 was identified by SDS-PAGE and analyzed by optical density scanning. The results showed that the purity was over 95%. The results of N-terminal amino acid sequence analysis showed that the first 15 amino acid sequences of the N-terminal of the rhIL-15 were consistent with the expected ones. Capillary electrophoresis analysis showed that the purity of purified rhIL-15 was close to 100 and the isoelectric point was 4.14, which was close to the theoretical value of the isoelectric point of rhIL-15 (4.42). The proliferative activity of purified rhIL-15 in CTLL-2 cells was 6.7 脳 10 ~ (-1) U / mg. Therefore, it can be determined that the expressed protein isolated and purified is rhIL-15..
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392;R730.5
本文编号：2397854
[Abstract]:Interleukin-15 (IL-15) is a cytokine with immunomodulatory activity discovered by Grabstein et al in 1994. It has biological functions similar to that of interleukin-2 (IL-2) and can promote T cells and B cells. The proliferation and differentiation of natural killer cell (NK), the enhancement of cytotoxic activity of cytotoxic T cell (CTL) / NK cells, etc. On the basis of our previous work, recombinant human interleukin-15 (rhIL-15) with purity of more than 95% was obtained by genetic engineering and protein purification techniques, and the mouse tumor model was established. The anti-tumor effect of rhIL-15 was studied, which laid a foundation for the development of rhIL-15 as a new anti-tumor drug. The main contents of this thesis are as follows: (1) prokaryotic expression, purification and identification of rhIL-15; (2) preparation of monoclonal antibody against rhIL-15; (3) acute toxicity test and antitumor effect of rhIL-15. (1) prokaryotic expression of rhIL-15, purification and identification of amino acid sequence of human interleukin-15 (hIL-15) mature protein, without changing amino acid sequence, Oligonucleotide fragments were synthesized by Escherichia coli codon preference, and hIL-15 gene was successfully synthesized by splicing PCR, and inserted into prokaryotic expression vectors pGEX-2T,pET_ (30a), pET_ (42), pETBlue-1 or pBV_ (220), respectively. It was expressed in a variety of host bacteria. Finally, the engineering strain BL_ (21) Star~ (TM) (DE3) plysS/pBV_ (220)-IL-15. was screened to express rhIL-15 stably and efficiently. In the engineering strain BL_ (21) Star~ (TM) (DE3) plysS/pBV_ (220)-IL-15, rhIL-15 was expressed as inclusion body, and the highest expression amount was 15.2g of the total cell protein. Western blot analysis showed that the rhIL-15 could specifically bind to monoclonal antibody against hIL-15. After induced expression, the inclusion bodies were isolated, washed repeatedly and dissolved in 6mol/L guanidine hydrochloride to obtain rhIL-15 crude products. After being separated by reverse phase chromatography and dissolved in 8mol/L urea, the rhIL-15 with bioactive rhIL-15, was obtained by dialysis or dilution renaturation. The purified rhIL-15. was obtained by DEAE weak anion exchange chromatography and Sephadex G-25 desalination. The purified rhIL-15 was identified by SDS-PAGE and analyzed by optical density scanning. The results showed that the purity was over 95%. The results of N-terminal amino acid sequence analysis showed that the first 15 amino acid sequences of the N-terminal of the rhIL-15 were consistent with the expected ones. Capillary electrophoresis analysis showed that the purity of purified rhIL-15 was close to 100 and the isoelectric point was 4.14, which was close to the theoretical value of the isoelectric point of rhIL-15 (4.42). The proliferative activity of purified rhIL-15 in CTLL-2 cells was 6.7 脳 10 ~ (-1) U / mg. Therefore, it can be determined that the expressed protein isolated and purified is rhIL-15..
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392;R730.5
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