【作者】 张平涛；

【导师】 林祥志； 方宏清； 杨丰；

【作者基本信息】 厦门大学 ， 动物学， 2008， 硕士

【摘要】 羧肽酶原B由胰腺细胞分泌,起初以含信号肽及前肽的前羧肽酶原B(preprocarboxypeptidase B)形式存在,在转运至内质网的过程中被信号肽酶切除掉信号肽而形成无活性的羧肽酶原B(procarboxypeptidase B,proCPB),羧肽酶原B在小肠经胰蛋白酶特异水解剪切前肽后而被活化成羧肽酶B。羧肽酶B(carboxypeptidase B,CPB)是一类可水解蛋白或多肽底物C-端Lys或Arg的金属蛋白酶。羧肽酶B在科研上主要用于蛋白质、多肽的测序;在医学上用于诊断胰腺炎;另外,羧肽酶B是重组胰岛素的生产过程中不可缺少的一个工具酶。目前,商业途径获得的羧肽酶B主要从猪胰脏中提取,使得产品价格昂贵,同时也不可避免地混杂有其它蛋白酶,如胰蛋白酶,胰凝乳蛋白酶。除了从胰腺组织中直接提取外,羧肽酶B也可通过基因工程途径获得。Hartman等和Li等分别研究了鼠CPB在大肠杆菌中的原核表达。Eaton等和Ventura等曾在毕赤酵母(Pichia pastoris)中分别表达了人血浆羧肽酶原B和猪胰腺羧肽酶原B。王德解等利用巴斯德毕赤酵母pAOX1系统诱导表达了鼠羧肽酶原B,为探索利用GAP启动子(pGAP)取代AOX1启动子(pAOX1)的重组质粒在巴斯德毕赤酵母(Pichia pastoris)中组成型表达鼠羧肽酶原B的可能性,本实验试图利用巴斯德毕赤酵母pGAP系统组成型表达鼠羧肽酶原B。本实验中,根据GAP启动子目的基因的序列设计上下游引物,应用PCR技术以质粒pIB2为模板扩增了GAP启动子基因片段,在目的基因的两端分别引入SacⅠ和BamHⅠ两个酶切位点,将GAP启动子基因片段取代诱导型质粒pPIC9K-proCPB上的AOX1启动子基因片段构建组成型表达质粒pGAPK-proCPB。用BglⅡ线性化pGAPK-proCPB重组质粒后,经电击转化到巴斯德毕赤酵母GS115感受态细胞中,涂布MD平板,筛选出阳性菌落,在含0.2%的葡萄糖培养基中培养工程菌,首次实现了鼠羧肽酶原B在毕赤酵母中的组成型表达,并在α-因子信号肽的引导下成功地分泌到胞外。表达产物经过疏水、离子交换层析纯化后,获得了重组proCPB目的蛋白。Fibrolase是从美国南方铜头蛇——Agkistrodon contortrix contortrix的毒液中分离出来的一种锌金属蛋白酶;具有直接的纤溶活性,而无出血活性。Fibrolase由203个氨基酸残基组成,分子量23kDa,其完整的氨基酸序列已被确认,N-末端为环化的谷丙酰胺残基。Alfimeprase由201个氨基酸组成,是Fibrolase的N-末端突变体(前3个氨基酸EQR-被Ser取代)。这种改构避免Alfimeprase N-末端出现环化的谷氨酰胺残基。Alfimeprase与Fibrolase具有同样的溶栓功能,能专一降解纤维蛋白(原)。本实验中,采用巴斯德毕赤酵母表达系统表达该锌金属蛋白酶。根据人工合成的Alfimeprase目的基因的序列设计其上下游引物,应用PCR技术扩增目的基因,在Alfimeprase目的基因的两端分别引入XhoⅠ和EcoRⅠ两个酶切位点。将PCR产物插入pPIC9毕赤酵母表达质粒载体中,构建重组质粒pPIC9-ALF;用BglⅡ线性化pPIC9-ALF重组质粒后,经电击转化到巴斯德毕赤酵母GS115感受态细胞中,涂布MD平板,筛选出阳性菌落。用终浓度1%的甲醇诱导pPIC9-ALF/GS115工程菌,表达Alfimeprase目的蛋白。另外,在Alfimeprase多克隆抗体的制备实验中,采用工程菌pET22b-ALF/BL21(DE3)用含氨苄青霉素的FML培养基,经乳糖诱导表达Alfimeprase目的蛋白。培养的工程菌菌体经超声破碎后,离心,洗涤、溶解包涵体。将纯化后的包涵体溶解液用弗氏佐剂乳化后,免疫BalB/C小鼠;共免疫三次,其中加强免疫两次。通过尾部取血和眼球取血的方法,获取小鼠血液;离心,获取血清,采用Elisa法检测获得的血清的效价。更多还原

【Abstract】 Naturally occurring rat carboxypeptidase B is produced from a precursor protein, preprocarboxypeptidase B, containing a 108 amino acid long N-terminal extension, which included the signal sequence (13 amino acid) and an activation peptide (95 amino acid). During transport of preprocarboxypeptidase B to the endoplasmatic reticulum, the signal peptide is cleaved off; the resulting enzymatically inactive proCPB precursor is secreted from the cell. The active CPB is then formed by cleavage of the activation peptide by trypsin in small intestine.Carboxypeptidase B(CPB) (EC 3.4.17.2) is a metalloenzyme, which contains one zinc atom per molecule and selectively hydrolyzes Arginine and Lysine from the carboxyl terminus of protein or peptides.CPB has been widely used for commercial and research purposes now, such as in protein sequence analysis, C-terminal trimming of proteins and peptides, and in the diagnosis of acute pancreatitis. In the activation of proinsulin to insulin, CPB was one of the two necessary enzymes, trypsin and CPB. However, commercially available CPB purified from porcine pancreas was very expensive, and was not totally free of other proteases, such as trypsin and chymotrypsin.CPB also can obtained by the engineering methods except for purifying from pancreas.Hratman et al and Li et al published the expression and purification of rat proCPB in Escherichia Coli respectively.Eaton et al and Ventura et al cloned and expressed human plasma proCPB and porcine proCPB in the methylotrophic yeast Pichia pastoris.Wang D J et al published the expression and purification of SD rat proCPB in the methylotrophic yeast Pichia pastoris using the AOX1 promoter.We amplify the GAP gene promoter from plasmid pIB2,construction of constitutive expression vector and the pGAP controlled constitutive expression of procarboxypeptidase B in Pichia pastoris in order to evaluate its possibility as an alternative to the AOX1 promoter. In this experiment, the sense and anti-sense primes were designed according to the synthesized gene sequence of the GAP promoter. Sac I and BamH I enzyme sites were cloned in the 5’ and 3’ terminals.The GAP gene of promoter was amplified by PCR from plasmid pIB2 and used to replace the AOX1 promoter(pAOX1) on plasmid pPIC9K-proCPB . A recombinant plasmid pGAPK-proCPB was constructed. It was linearized with the enzyme BglⅡand transformed into the Pichia pastoris GS115 competent cell by electroporation, and spread on MD plate. Positive clones were screened by HIS4 selectable marker and PCR. The genetic engineered strain pGAPK-proCPB/GS115 was cultured in a medium with a final concentration of 0.2% glucose. Recombinant proCPB was successfully expressed in Pichia pastoris for the first time,and could be secreted into the culture supernatant .The recombinant proCPB was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Phenyl FF. Fibrolase, a metalloproteinase isolated from the venom of Agkistrodon contortrix contortrix (southern copperhead snake), is a direct-acting fibrin(ogen)olytic enzyme, but it is not hemorrhagic. The molecular weight of fibrolase is approximately 23,000 and the protein contains 203 amino acids in known sequence,with an N-terminal of the cyclized glutamine isoform formation.Alfimeprase is a mutant of Fibrolase at N-terminal sequence,contains 201 amino acids with an N-terminal sequence of SFPQR---,in contrast to the N-terminal sequence of fibrolase ,which begins with EQRFPQR---.The amino terminal modification in Alfimeprase solved the problem of the cyclized glutamine isoform formation.Alfimeprase can also promote thrombolysis by activation of the endogenous fibrinolytic system as well as Fibrolase. Alfimeprase has been demonstrated the proteolytic activity against fibrinogen .In this experiment, we try to express this metalloproteinase in the Pichia pastoris expression system, the sense and anti-sense primes were designed according to the synthesized gene sequence of the Alfimeprase. The gene of interest was amplified by PCR. Xho I and EcoR I enzyme sites were cloned in the 5’ and 3’ terminals. PCR production was connected to pPIC9 expression vector with T4 DNA ligase. A recombinant plasmid pPIC9-ALF was constructed. It was linearized with the enzyme BglⅡand transformed into the Pichia pastoris GS115 competent cell by electroporation, and spread on MD plate. Positive clones were screened by HIS4 selectable marker and PCR.The genetic engineered strain pPIC9-ALF/GS115 was induced with a final concentration of 1% methanol. Alfimeprase was expressed.In the preparation of the polyclonal antibody of Alfimeprase,the strain pET22b-ALF/BL21(DE3) genetically engineered was cultivated in L FML cultivation medium containing appropriate concentrate of ampicillin. It was induced withα-Lactose , and Alfimeprase was expressed. The collected E.coli cells were lysed using ultrasonic, and the aggregates collected by low-speed centrifugation. Inclusion bodies were washed and solubilized with strong protein denaturants, e.g. 6 mol/L urea. The solubilized inclusion bodies were mixed and emulsified with Freund’s adjuvant. The solution was used to immunize BalB/C rats for three times. The last twice immunizations were strength. To get the serum of BalB/C rats, blood was obtained from tail and eyes and centrifuged at a low speed. The titer of the polyclonal antibody of Alfimeprase was identified by Elisa.更多还原