人脂联素球状结构域（gAd）基因的克

发布时间：2019-01-04 10:34

【摘要】：目的构建gAd 原核表达体系并对培养条件进行优化,对目的蛋白分离、纯化、复性并测定其生物活性;同时构建gAd 融合蛋白表达体系,完成对融合蛋白的纯化。方法本研究首先分离健康人外周血淋巴细胞,用酚-氯仿法抽提基因组DNA,从Genbank 获取脂联素的基因序列,用Oligo 6.44 软件设计引物,使其扩增产物中包含脂联素球状结构域基因全部序列,PCR 产物用低熔点琼脂糖凝胶切胶回收纯化,纯化的扩增产物和T 载体的连接,连接产物转化JM109感受态细胞,在氨苄阳性平板中筛选白色菌落,扩种后提取质粒用酶切和PCR 鉴定,该质粒命名为pMD18T-Ad。以pMD18T-Ad 为模板,设计合适引物,在上游引物中加入EcoR I 酶切位点和起始密码,在下游引物中加入终止密码和Pst I 酶切位点,把上述扩增产物经切胶回收纯化后与pBV220 经EcoR I 和Pst I 双酶切纯化后的产物连接,把该质粒命名为pBV220-gAd,转化大肠杆菌DH5α,筛选出阳性菌株,并经测序进一步证实,温控诱导表达,超声破菌,包涵体的分离洗涤纯化复性。复性后用超滤管离心浓缩,0.22μm 微孔滤膜过滤除菌,用考马斯亮蓝法测定蛋白含量,把一定量的gAd注入高糖和高脂肪酸的兔体内,不同时间采血观察其降低血糖和游离脂肪酸的效果。以pBV220-gAd 为模板通过PCR 方法构建C 端含有His Tag 的pBV220-gAdhis 质粒并转化大肠杆菌DH5α,用Western-blotting 鉴定,用Ni~(2+)-NTA 树脂柱对对表达产物进行纯化。
[Abstract]:Objective to construct a prokaryotic expression system of gAd and optimize the culture conditions to isolate, purify, refolding and determine its biological activity. At the same time, the expression system of gAd fusion protein was constructed, and the fusion protein was purified. Methods in this study, the peripheral blood lymphocytes of healthy people were isolated. The gene sequence of adiponectin from Genbank was extracted by phenol-chloroform method. Primers were designed with Oligo 6.44 software. The amplified product contained all the adiponectin globular domain genes. The PCR product was recovered and purified by low melting point agarose gel. The purified product was connected with T vector and transformed into JM109 receptive cells. White colonies were screened from positive plates of ampicillin. The plasmids were digested by enzyme and identified by PCR. The plasmid was named pMD18T-Ad.. Using pMD18T-Ad as template, suitable primers were designed, EcoR I restriction site and start code were added to upstream primer, and stop codon and Pst I restriction site were added to downstream primer. The above amplified products were recovered and purified by gelling gel and ligated with the products of pBV220 digested by EcoR I and Pst I. The plasmid was named pBV220-gAd, and transformed into Escherichia coli DH5 伪. The positive strains were screened out and further confirmed by sequencing. Temperature control induced expression, ultrasonic bacteriostasis, purification and renaturation of inclusion bodies. After renaturation, ultrafiltration tube was used to centrifuge concentration, 0.22 渭 m microporous membrane was used to filter bacteria, Coomassie brilliant blue method was used to determine protein content, and a certain amount of gAd was injected into rabbit with high sugar and high fatty acid. Blood samples were collected at different times to observe the effect of reducing blood glucose and free fatty acids. Using pBV220-gAd as template, pBV220-gAdhis plasmid containing His Tag in C terminal was constructed by PCR method and transformed into E. coli DH5 伪. The expression product was purified by Ni~ (2)-NTA resin column and identified by Western-blotting.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78

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