牦牛β-防御素5基因的原核表达及其乳腺特异性表达载体的构建.doc

摘要 本研究以牦牛β-防御素5基因为研究对象，分别对牦牛β-防御素5基因进行原核和真核表达研究。原核表达以pET-32a(+)为表达载体，将构建好的原核表达载体转化BL21（DE3）进行表达，检测重组蛋白的体外抑菌活性。真核表达以牛β-酪蛋白基因（β-casein，CSN2）5 端和3 端为调控成分，牦牛β-防御素5 为表达序列，构建牦牛β-防御素5乳腺特异性表达载体，将表达载体导入体外培养的牦牛乳腺上皮细胞，分析牦牛β-防御素5基因的表达效率。具体如下： 采用RT-PCR方法从牦牛肺组织中扩增牦牛BNBD5基因成熟肽编码及BNBD5开放阅读框。PCR产物经胶回收纯化后，克隆于pMD 19-T载体的T位点。测序结果表明，获得了138 bp 的BNBD5成熟肽编码片段及29 bp的开放阅读框，成熟肽编码45个氨基酸残基，内含6个位置保守的半胱氨酸残基。经遗传进化分析发现，该基因的序列与牛的mRNA同源性最高，可达到86.%。 将重组表达质粒转化大肠杆菌BL21，于37 °C 0?6 h间进行诱导表达，SDS检测融合蛋白表达情况，并对该重组蛋白进行纯化测定其体外抗菌活性。结果表明，经IPTG诱导，获得了分子量约为25 kDa的牦牛β防御素5成熟肽融合蛋白。琼脂糖扩散法表明，0.08 mg/mL的纯化蛋白对大肠杆菌及金黄色葡萄球菌有抗菌作用。 利用普通PCR法扩增了奶牛CSN2 5 端5不翻译区的第一个外显子）、3 端（）调控区及牦牛β-防御素5基因的开放阅读框，并利用双酶切定向克隆的方法，将他们克隆于pEGFP-C1真核表达载体上，构建了牦牛β-防御素5乳腺特异性表达载体。 采用脂质体转染的方法将构建的牦牛β-防御素5乳腺特异性表达载体导入体外培养的牦牛乳腺上皮细胞中，经新霉素抗性基因（G418）初步筛选后，借助荧光显微镜观察到了绿色荧光蛋白（GFP）基因的表达。通过设计一对特异性引物，对初步筛选的阳性克隆进行检测，结果表明，表达载体已整合到乳腺上皮细胞的染色体中，证明了构建的乳腺特异性表达载体的可行性。  Abstract The object of this study was yak beta-defensin 5 (BNBD5), which prokaryotic and eukaryotic expression was studied respectively. Prokaryotic expression was used pET-32a (+) as vector, then transformed the constructed prokaryotic expression vector into BL21 (DE3) and detected the antibacterial activity of recombinant protein in vitro. The mammary gland-specific expressional vector of yak BNBD5, which was partial consisted of the 5′and 3′regulation regions of cow β-casein, was constructed with cDNA of yak BNBD5. In order to study the rationality of expression vector，the vector was transfected into mammary epithelial cells. The results of this research as following: The mature peptide encoding region and open reading frame of yak BNBD5 were cloned from lung tissues by RT?PCR. After PCR, products were recovered and purified, the aim fragments were cloned at T site of pMD 19-T Vector. The results of sequence analysis indicated the mature peptide encoding region and open reading frame of yak BNBD5 were 138 bp and 209 bp, respectively. The encoding region encodes a polypeptide of 45 amino acids and has six conserv