【摘要】：白細(xì)胞介素-15(IL-15)是Grabstein等人于1994年發(fā)現(xiàn)的一種具有免疫調(diào)節(jié)活性的細(xì)胞因子，它具有與白細(xì)胞介素-2(IL-2)相似的生物學(xué)功能，可促進(jìn)T細(xì)胞、B細(xì)胞、自然殺傷細(xì)胞(NK)的增殖與分化，增強細(xì)胞毒T細(xì)胞(CTL)／NK細(xì)胞的細(xì)胞毒活性等，并能發(fā)揮優(yōu)于IL-2的抗腫瘤作用。 本論文是在我們的前期工作基礎(chǔ)上，通過基因工程技術(shù)和蛋白質(zhì)純化技術(shù)獲得了純度在95％以上的重組人白細(xì)胞介素-15(rhIL-15)，并用建立的小鼠腫瘤模型，研究了rhIL-15的抗腫瘤作用，為將rhIL-15開發(fā)成一類抗腫瘤新藥奠定了基礎(chǔ)。本論文工作主要研究內(nèi)容包括三部分：(1)rhIL-15的原核表達(dá)、純化和鑒定；(2)抗rhIL-15單克隆抗體的制備；(3)rhIL-15的急性毒性實驗和抗腫瘤作用研究。 (1)rhIL-15的原核表達(dá)、純化和鑒定 根據(jù)已知人白細(xì)胞介素-15(hIL-15)成熟蛋白的氨基酸序列，，在不改變氨基酸序列的前提下，采用大腸桿菌偏愛密碼子合成寡核苷酸片段，通過拼接PCR法成功合成hIL-15基因，分別插入原核表達(dá)載體pGEX-2T、pET_(30a)、pET_(42)、pETBlue-1或pBV_(220)，在多種宿主菌中獲得了表達(dá)，最后篩選到穩(wěn)定高效表達(dá)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以包涵體形式表達(dá)，最高表達(dá)量為菌體總蛋白的15.2％。經(jīng)蛋白印跡法鑒定表明，此rhIL-15能與抗hIL-15的單克隆抗體特異結(jié)合。誘導(dǎo)表達(dá)后，分離出包涵體，經(jīng)反復(fù)洗滌后，溶于6mol／L鹽酸胍，獲得rhIL-15粗品。再經(jīng)反相層析分離后溶于8mol／L尿素，經(jīng)透析或稀釋復(fù)性獲得有生物活性的rhIL-15，復(fù)性后的rhIL-15依次經(jīng)DEAE弱陰離子交換層析和Sephadex G-25脫鹽得到純化的rhIL-15。純化的rhIL-15行SDS-PAGE鑒定及光密度掃描分析，結(jié)果顯示，純度在95％以上。N端氨基酸序列分析結(jié)果表明，此rhIL-15 N端的前15個氨基酸序列與預(yù)期的一致；毛細(xì)管電泳分析表明，純化rhIL-15的純度接近100％，等電點為4.14，接近rhIL-15的等電點理論值4.42。純化rhIL-15的體外促CTLL-2細(xì)胞增殖活性為6.7×10~6U／mg。因此，可以確定分離純化的表達(dá)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..
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R392;R730.5

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