摘要
本论文以重要的海水养殖经济鱼大菱鲆为实验材料,采用分子克隆与体外重组技术,对影响鱼类生长和抗病的重要基因胰岛素样生长因子-I(IGF-I)、抗菌肽hepcidin与IGF-I的重组基因hep-IGF、CXC趋化因子S457和CC趋化因子KC70进行了克隆和原核表达的研究,并采用不同的活性测定法对几种蛋白表达产物进行了活性研究。
1.大菱鲆胰岛素样生长因子-I的克隆、原核表达及促生长活性分析
通过RT-PCR方法从大菱鲆肝组织克隆了胰岛素样生长因子-I(IGF-I)成熟肽片段,分析表明此成熟肽由70个氨基酸残基组成,含有3个链内二硫键。经序列比对,发现大菱鲆IGF-I是高度保守的蛋白,其成熟肽的氨基酸序列与牙鲆、金鲈、草鱼、鸡、人的相似性分别为100%、98%、86%、79%、76%。将扩增片段克隆到原核表达载体pGEX-4T-1上,实现了IGF-I成熟肽和GST蛋白在E.coli BL21(DE3)plysS中的的融合表达。融合蛋白分子量约为34kD,诱导4h时占菌体总蛋白的59%,主要以包涵体形式存在。Western-blotting免疫印迹表明融合蛋白可以特异性地被anti-GST抗体识别。对菌体进行超声破碎,收集包涵体并用1%Triton-X100+1%DOC洗涤,经6mol/L盐酸胍变性溶解及脉冲法稀释复性(0.5 mol/L L-Arginine, 1.0 mol/L GSH,0.2 mol/LGSSG.)后,通过GSTrap FF亲和预装柱纯化,获得了电泳分析纯的融合蛋白。以细胞增殖实验检测蛋白生物活性,结果显示纯化蛋白能促进大菱鲆肾脏细胞的增殖。
2.大菱鲆融合蛋白hepcidin-IGF的重组表达及活性分析
以实验室构建的pMD-hep-IGF质粒为模板进行PCR,在片段上添加EcoR I和Xho I酶切位点。经过酶切、连接和转化,将扩增的hep-IGF片段克隆至pGEX-4T-1重组表达载体,并转化E.coli BL21(DE3)plysS宿主菌。阳性重组克隆用IPTG进行不同时间的诱导,结果表明hep-IGF重组蛋白在大肠杆菌中获得了高效表达。随着诱导时间的增加,重组蛋白的表达量增加,在诱导后7h时达最大,大约占大肠杆菌总蛋白的55%。western-blotting免疫印迹表明,重组菌所表达的融合蛋白可以特异性地被anti-GST抗体识别。对菌体进行超声破碎,收集上清过GSTrap FF亲和柱,获得了纯化的GST-hep-IGF重组蛋白。用液体抑制法验证hep-IGF融合蛋白的抑菌活性,结果表明大肠杆菌(ATCC25922)培养8h后,加重组蛋白的处理组与不加任何样品的处理组相比出现抑制效果。
3.大菱鲆趋化因子S457和KC70的原核表达及趋化活性分析
以大菱鲆肝脏cDNA为模板进行PCR,获得了S457和KC70成熟肽片段。序列比对发现这两种趋化因子与其他物种的CXC或CC趋化因子的相似性不高:大菱鲆S457与斑马鱼CXCL-C1c、鼠CXC L7、猪CXC L9、人CXC L5相似性分别为34%、39%、42%、39%;大菱鲆KC70与大黄鱼CC样趋化因子、大西洋鲑CCL19、斑马鱼CCL-C5a、鸡CCL19、人CCL19相似性分别为61%、39%、35%、45%、38%。将扩增片段克隆到原核表达载体pGEX-4T-1上,实现了融合表达。GST-S457融合蛋白分子量约为38.6kD,诱导4h时表达量最大,占菌体总蛋白的47.5%;GST-KC70融合蛋白分子量约为36.1kD,诱导4h时表达量最大,占菌体总蛋白的45.6%。Western-blotting免疫印迹表明融合蛋白可以特异性地被anti-GST抗体识别。对菌体进行超声破碎,收集上清过GSTrap FF亲和柱,纯化的蛋白进行趋化活性分析。结果显示当GST-S457重组蛋白浓度为0.1和1ug/mL时,与对照组相比具有显著差异(P<0.01),且后者趋化作用更明显;当GST-KC70重组蛋白浓度为10ug/mL时,与对照组相比具有显著差异(P<0.01)。
Based on the molecular cloning and recombination techniques, 4 immunity-related or growth-related genes (IGF-I, hep-IGF, CXC-S457 and CC-KC70) from turbot were cloned to the pGEX-4T-1 vector and then transformed into E.coli BL21(DE3)plysS. Induced by IPTG, the recombinant proteins expressed and then were purified with GSTrap FF affinity chromatograph. The biological activities of the proteins were also analyzed with different methods.
1. Cloning, recombinant expression and bioactivity analysis of Turbot IGF-I
Insulin-like growth factor-I (IGF-I) is a conserved peptide expressed ubiquitously, which shows diverse effects on development, growth, and metabolism. With RT-PCR, the fragment encoding the Turbot (Scophthalmus maximus) mature IGF-I peptide was amplified. It was predicted that the mature peptide was composed of 70 amino acids including 6 cysteines which could form 3 disulfide bonds. The target fragment was successfully subcloned into the expression vector pGEX-4T-1, after induction with IPTG, the fusion proteins highly expressed in E.coli BL21(DE3)plysS. The result of SDS-PAGE showed that the fusion protein expressed in the form of inclusion bodies with molecular weight of 34 kD and maximally amounted to 59 % of the whole protein in the E.coli cell 4hours post induction. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. The inclusion bodies were dissolved in 6 mol/L guanidine chloride followed by pulse renaturation in refolding buffer containing 0.5 mol/L L-Arginine, 1.0 mol/L GSH and 0.2 mol/L GSSG. Then the renatured recombinant protein was purified by GSTrap FF affinity chromatography. The effect of purified GST-IGF-I on turbot kidney cells was analysised, and it indicated that the recombinant GST-IGF-I can stimulate the proliferation of the cells.
2. Recombinant expression and bioactivity analysis of Turbot hep-IGF-I
The hep-IGF recombinant gene was amplified using a pair of specific primers. The PCR products were cloned to pGEX-4T-1 vector and then the plasmid was transformed into E.coli BL21(DE3)plysS. After induced by IPTG for different time courses, the expression of GST-hep-IGF was determined by SDS-PAGE. The result shows that the expression amont can raised to the peak 7 hours post induction, maximally amounted to 55 % of the total protein in the E.coli cell. Western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. After ultrasonic disruption process, the supernatant was collected and pumped into GSTrap FF affinity Collumn. The antibacterial bioactivity of purified recombinant proteins to E.coli (ATCC25922) was analyzed with liquid growth inhibition method, and the different antibacterial effects were detacted in 8 hours post treatments.
3. Recombinant expression and bioactivity analysis of CXC chemokine S457 and CC chemokine KC70 from Turbot
The S457 and KC70 mature genes were obtained with PCR using turbot liver cDNA as template. Sequence blast showed that the two chemokines shared low identity and similarity, that is about 30~60% identities with other chemokines of several animals. The amplified segments were subcloned to the pGEX-4T-1 vector respectively. After transformation and induction, the expression of recombinant proteins were detected with SDS-PAGE and the molecular weights of GST-S47 and GST-KC70 recombinant proteins were about 38kD and 36kD. The result of time course experiment showed the amount of GST-S47 and GST-KC70 reached the maximums 4 hours post induction, accounted to 47% and 46%, respectively. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. After ultrasonic disruption process, the supernatant is collected and pumped into GSTrap FF affinity Column. Then a chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5um pores towards GST-S47 and GST-KC70 at the optimal concentration of 1ug/mL and 10ug/mL respectively.
引文
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