【作者】 陈建梁；

【导师】 关怡新；

【作者基本信息】 浙江大学 ， 生物化工， 2005， 硕士

【摘要】 凝血酶是目前应用最为广泛的一种止血剂,在临床上有非常重要的用途。凝血酶原-2(pThr-2) 是α-凝血酶生成过程中的分子量最小的单链中间前体,经激活后可转化为有活性的α-凝血酶。本论文通过将含凝血酶原-2基因的质粒克隆至大肠杆菌中并得到大量表达,但这样获得的凝血酶原为包涵体形式,必须经过体外复性后,再激活为凝血酶。本文在优化发酵条件获得高产量的重组牛凝血酶原-2包涵体的基础上,对牛凝血酶原-2的体外重折叠复性如稀释复性以及温敏型聚N-异丙基丙烯酰胺(PNIPA)凝胶和二硫键异构酶(DsbA)介导的复性进行了详细的研究。将携带已克隆pThr-2基因的质粒成功地转化到大肠杆菌BL21(DE3) 中,经IPTG诱导后继续培养,获得凝血酶原-2包涵体。对培养基组成及操作条件进行了优化,详细考察了碳源、氮源、无机盐、诱导起始时间、诱导剂添加量、接种量、发酵温度和转速等因素对目标蛋白表达量的影响。最终在优化的培养条件下,重组凝血酶原-2包涵体的产量为110 mg/L发酵液,占菌体总蛋白的21. 7%。将收集的菌体用超声波破碎后,离心得到pThr-2包涵体,然后Tris-HCl缓冲液洗涤包涵体,并优化了洗涤液组成。包涵体用8 M尿素(含0. 3 M DTT)溶解后,在单因素实验的基础上,采用一次回归正交试验及快速登高试验,综合考察了pThr-2包涵体稀释复性过程的关键参数,包括GSSG浓度、GSSG/GSH比例、尿素浓度、PEG6000浓度、L-Arg添加量以及复性温度等。凝血酶活力用生色底物法测定,结果在优化条件下凝血酶活力可达2948U/mL。在稀释复性的基础上,进一步考察了添加温敏型PNIPA凝胶和DsbA协助凝血酶原-2体外复性的效果。实验结果表明,加入凝胶协助复性后凝血酶活力可达6222 U/mL,凝胶的最佳添加量为105 mg/mL,且复性初始蛋白浓度越高,凝胶协助复性的效果就越明显;但PNIPA凝胶的加入在协助复性的同时,也在一定程度上延长了复性达到平衡所需的时间。我们将含有DsbA基因的pAVD63质粒转化到大肠杆菌中,并在优化的培养条件下发酵制备DsbA,经离子交换层析获得的高纯度DsbA用于协助pThr-2复性。由实验结果看出,添加DsbA能够显著改善凝血酶原-2的复性效果,当DsbA与pThr-2的摩尔比为4:1时,凝血酶活力相对稀释复性提高了41. 7%。凝血酶经肝素亲和柱纯化后,荧光光谱分析表明复性后经激活得到的凝血酶具有与天然态凝血酶相同的最大荧光发射波长,表明凝血酶原-2已正确折叠。更多还原

【Abstract】 Thrombin is one of the most widely used hemostats. Prethrombin-2 (pThr-2), the smallest single-chain precursor of thrombin, may be activated to thrombin by snake venom. Thrombin is mostly isolated from plasma of animals. pThr-2 gene was cloned to E. coli to produce recombinant prethrombin-2, which was formed as inclusion bodies and must be refolded to obtain native conformation. After pThr-2 inclusion bodies were produced efficiently in the optimized culture conditions, we studied the refolding of pThr-2 in direct dilution process, and further with the assistant of Poly(N-isopropyl acrylamide)(PNIPA) hydrogel and disulfide binding protein A(DsbA).Vector with gene of pThr-2 was successfully transformed into E. coli BL21 (DE3). After the cells were induced by IPTG and further cultured, pThr-2 inclusion bodies were obtained. The fermentation conditions, such as culture medium, IPTG concentration, fermentation temperature, and shake speed for the pThr-2 production were optimized. The inclusion bodies of the pThr-2 produced in the E. coli culture achieved 110 mg/L, accounting for 21.7% of the total cell proteins.After harvested by centrifuge and washed by Tris-HCl buffer(consisting of EDTA, urea and Triton X-100) and solubilized by 8M urea and 0.3M DTT, the key parameters in the refolding process, such as GSSG concentration, GSSG/GSH, urea concentration, L-Arg concentration and operation temperature were investigated by linear regression orthogonal experiments combined with steepest ascent experiments. Good results were obtained under optimized conditions. The total activity of thrombin assayed using chromogenic and fluorogenic substrate was about 2948U/mL at optimum refolding process.It was observed that PNIPA and DsbA were quite efficient in assisting the renaturation of bovine pThr-2. When mixing with 105mg/mL PNIPA hydrogel during the refolding, the thrombin activity was 6222U/mL, which was much higher than that refolded by direct dilution, but the time required for the refolding with PNIPA was a little longer than that by dilution. pAVD63 vector with gene of DsbA was successfully transformed into E. coli to produce DsbA in optimized culture conditions, which was purified by ion-exchange chromatography and used to assist the refolding of pThr-2. When the concentration ratio of DsbA to protein was 4 : 1, the gained thrombin activity was 41.7% higher than that by simple dilution.The thrombin was purified by heparin affinity chromatography for fluorescence spectrum experiments, which indicated the thrombin activated from the refolded pThr-2 had the same emission wavelength with native thrombin.更多还原