副猪嗜血杆菌hxuCBA基因簇克隆表达及其免疫特性研究

发布时间：2018-05-22 16:10

本文选题：副猪嗜血杆菌 + hxuCBA　；参考：《四川农业大学》2015年硕士论文

【摘要】：由副猪嗜血杆菌(Haemophlius parasuis, HPS)引起的Glasser's'病已经给全球养猪业造成重大损失。铁几乎是所有生物体不可或缺的微量营养元素,它可以作为多种生物过程的电子受体或供体发挥作用。血红素是细菌重要的铁源之一,由hxuCBA基因簇组成的hxu血红素摄取系统是副猪嗜血杆菌获取铁源的途径之一。本文通过对组成该系统的三个基因进行克隆表达,并分别对表达产物进行了生物信息学分析、免疫原性、免疫保护性、抗血清杀菌等研究。主要研究结果如下：1.HPS hxuCBA基因簇的克隆、鉴定及分析针对HPS hxuC、hxuB、hxuA基因(组成hxu血红素摄取系统的基因)分别设计一对引物(各基因片段中信号肽序列已去除),基因片段大小分别为2064bp、 1617bp、2847bp。通过PCR的方法从HPS四川M-3分离株的基因组中扩增得到hxuC、hxuB、hxuA基因,分别构建pMD19-T-C、pMD19-T-B、pMD19-T-A重组克隆质粒,三种质粒经测序鉴定显示突变碱基未形成终止密码子,与HPS SH0165菌株的核苷酸序列一致性分别达99.22%、99.13%、98.11%,hxuC和hxuB基因序列无碱基缺失,hxuA基因序列有6个碱基缺失。对HPS HxuC、HxuB、HxuA氨基酸序列进行生物信息学分析,结果显示HxuC、HxuB、HxuA成熟期蛋白分别由688、539、949个氨基酸组成,蛋白大小分别为78.07kDa、60.72kDa、105.72kDa,等电点分别为9.23、9.34、6.36,均无跨膜区存在。三种蛋白的二级结构均由无规则卷曲(30.81%、30.80%、38.78%)、延伸链(19.48%、28.76%、29.08%)、α螺旋(40.55%、28.57%、22.13%)和p转角(9.16%、11.87%、10.01%)组成。HxuC口HxuB属于外膜蛋白,HxuA属于胞外蛋白。通过功能位点预测分析,三种蛋白均含有多个糖基化、磷酸化位点,另外HxuC、HxuB、HxuA分别含有1个TonB依赖受体蛋白信号2位点、1个ATP/GTP结合位点基序A(P环)、1个内质网定位序列。抗原表位分析显示HxuC、HxuB、HxuA蛋白均含有多个抗原决定簇位点,并且分别有30、20、38个与细胞毒性T细胞结合的抗原表位位点。同时对HxuC进行同源建模,发现HxuC蛋白在同种间同源性很高,而在同属与同科间同源性很低。2. HPS hxuCBA基因簇的表达及纯化利用BamHI和Ncol对pMD19-T-C、pMD19-T-B、pMD19-T-A和pET39b进行双酶切,16℃进行连接,构建pET39-C、pET39-B、pET39-A重组表达质粒,将重组表达质粒转入E.coil(BL21)表达菌内进行诱导表达。优化蛋白表达条件的结果显示,HxuC经37℃,0.2 mmol/L IPT G浓度诱导6 h达到最大表达量,HxuB经37℃,1.2mmol/L IPTG浓度诱导3 h达到最大表达量,HxuA在30℃,IPTG诱导浓度0.8mmol/L诱导2 h表达量最大。经SDS-PAGE显示三种重组蛋白均可以可溶性和包涵体形式存在,分子量大约为105kDa、87kDa、130 kDa(含载体部分)。然后通过镍离子亲和层析法纯化得到目的蛋白,纯化的蛋白经透析后进行western-blot结果表明三种蛋白均具有良好的反应原性。3.HPS HxuC、HxuB、HxuA蛋白的免疫特性研究以昆明小鼠作为实验动物,用自制的HxuC、HxuB、HxuA、HxuCBA (包含HxuC、 HxuB、HxuA)疫苗和HPS M-3全菌灭活苗进行皮下注射免疫。一免后第14天进行二免。一免和二免分别用弗氏完全佐剂和弗氏不完全佐剂。并分别用佐剂和PBS作为对照。每次免疫后7天对小鼠进行断尾收集血清。通过ELISA的方法检测抗体水平和细胞因子水平,结果显示小鼠抗1HxuC、HxuB、HxuA、HxuCBA、M-3血清的抗体效价分别达到1：6400、1：3200、1：1600、1：12800、1：12800；相比于免疫前,细胞因子IL-2、IL-4、IFN-γ的水平均有所升高,而且一免和二免结果呈现递增趋势。本研究进行了抗血清杀菌实验,结果显示HxuC、HxuB、HxuA、HxuCBA、M-3、佐剂、PBS组的细菌存活率分别为28.2%、32.3%、38.6%、35.1%、28.5%、46.9%、48.3%。由此可见,HxuC、HxuB、HxuCBA 对 HPS M-3有明显的抗菌作用,而HxuA的抗菌作用相对较弱。以昆明小鼠作为实验动物,测出HPS M-3菌株的半数致死量LD50,约为1.3×109cfu。随后进行了攻毒保护试验,免疫程序同上。另外设立全菌灭活免疫组。二免后第7天对小鼠进行腹腔攻毒(5×LD50),攻毒后统计小鼠发病死亡状况。结果显示PBS对照组和佐剂对照组小鼠全部死亡,HxuC、HxuB、HxuA、 HxuCBA、M-3免疫组保护率为87.5%、62.5%、37.5%、75.0%、87.5%。另外,这三种蛋白均能减少副猪嗜血杆菌对小鼠的组织损伤。这些研究表明HxuC、HxuB、 HxuCBA可作为亚单位疫苗的有效候选蛋白。
[Abstract]:Glasser's'disease, caused by Haemophlius parasuis (HPS), has caused great loss to the global pig industry. Iron is almost an indispensable micronutrient of all organisms. It can act as an electron acceptor or donor in a variety of biological processes. Hemoglobin is one of the important iron sources of bacteria, and the hxuCBA gene is one of the most important sources of the bacteria. The cluster of hxu heme uptake system is one of the ways to obtain the iron source of Haemophilus accessory. In this paper, three genes are cloned and expressed in this paper, and the bioinformatics analysis, immunogenicity, immunogenicity, antiserum killing bacteria and other studies are carried out respectively. The main results are as follows: 1.HPS hxuCBA Cloning, identification and analysis of the gene cluster, a pair of primers were designed for HPS hxuC, hxuB, hxuA gene (the gene of the hxu heme uptake system), respectively. The sequence size of the gene fragment was 2064bp, 1617bp, and 2847bp. was amplified from the genome of HPS Sichuan M-3 isolate by PCR. UB, hxuA gene, respectively, to construct pMD19-T-C, pMD19-T-B, pMD19-T-A recombinant cloned plasmids. The three plasmids were sequenced to show that the mutation base did not form a terminating codon. The nucleotide sequence of the HPS SH0165 strain was consistent with the nucleotide sequence of the HPS SH0165 strain, and the sequence of the hxuC and hxuB genes was missing, and the hxuA gene sequence had 6 base deletion. PS HxuC, HxuB, HxuA amino acid sequences were analyzed by bioinformatics. The results showed that HxuC, HxuB, HxuA mature proteins were composed of 688539949 amino acids respectively, the size of the protein was 78.07kDa, 60.72kDa, 105.72kDa, and the isoelectric points were respectively 9.23,9.34,6.36, and the two structure of the three proteins were all curled by irregular (30.81%). 30.80%, 38.78%), extension chain (19.48%, 28.76%, 29.08%), alpha helix (40.55%, 28.57%, 22.13%) and P corner (9.16%, 11.87%, 10.01%) are composed of.HxuC HxuB belonging to outer membrane protein, HxuA belongs to extracellular protein. Through functional site prediction analysis, three proteins contain many glycosylation, phosphorylation sites, and HxuC, HxuB, HxuA contain 1 TonB dependence respectively. The 2 site of receptor protein signal, 1 ATP/GTP binding sites A (P ring), 1 endoplasmic reticulum location sequences. Antigen epitope analysis shows that HxuC, HxuB, HxuA proteins contain multiple antigen determinant sites, and there are 30,20,38 antigen epitopes binding to cytotoxic T cells respectively. Meanwhile, the homologous modeling of HxuC is also found in the HxuC protein. Homologous homology is very high, and the expression and purification of.2. HPS hxuCBA gene clusters with low homology between the same family and the same family are expressed and purified using BamHI and Ncol for pMD19-T-C, pMD19-T-B, pMD19-T-A and pET39b to be double enzyme cut at 16 degrees C to construct pET39-C, pET39-B, pET39-A recombinant plasmid, and the recombinant expression plasmid is transferred into the expression bacteria. The optimum protein expression conditions showed that the maximum expression of HxuC was reached at 37 C, 0.2 mmol/L IPT G concentration induced 6 h, HxuB was 37, 1.2mmol/L IPTG concentration induced 3 h to reach the maximum expression, HxuA was 30, IPTG inducible concentration induced the maximum expression of 2, which showed that three recombinant proteins could all be available. The solubility and inclusion body form exist, the molecular weight is about 105kDa, 87kDa, 130 kDa (containing the carrier part). Then the target protein is purified by the nickel ion affinity chromatography. The purified protein has been dialysated for Western-blot results. The results show that all the three proteins have good reactive proto.3.HPS HxuC, HxuB, HxuA protein. Kunming mice, as experimental animals, were immunized subcutaneously with homemade HxuC, HxuB, HxuA, HxuCBA (including HxuC, HxuB, HxuA) vaccines and HPS M-3 whole bacteria inactivated vaccine. Two free and two free Freund and two Freund incomplete adjuvant were used for the first fourteenth days after immunization. The adjuvant and PBS were used respectively as control. 7 days after each immunization. The antibody level and the level of cytokine were detected by ELISA method. The results showed that the antibody titers of 1HxuC, HxuB, HxuA, HxuCBA, M-3 serum were respectively 1:6400,1:3200,1:1600,1:12800,1:12800, and the levels of IL-2, IL-4, IFN- gamma were higher than those before immunization. The results showed that the bacterial survival rate of HxuC, HxuB, HxuA, HxuCBA, M-3, adjuvant and PBS group was 28.2%, 32.3%, 38.6%, 35.1%, 28.5%, 46.9%, 48.3%., HxuC, HxuB, HxuCBA had obvious antibacterial effect on HPS M-3, and the antibacterial effect was relative to those of HPS M-3. With Kunming mice as experimental animals, the half lethal dose LD50 of HPS M-3 strain was measured, about 1.3 x 109cfu. followed by the attack protection test. The immunization program was the same. In addition, the whole bacteria inactivation immunization group was set up. The mice were attacked by abdominal attack (5 * LD50) after seventh days of exemption. After attack, the death situation of mice was statistically analyzed. The results showed PBS pairs. All the mice in the group and the adjuvant control group died, HxuC, HxuB, HxuA, HxuCBA, and M-3 immune groups were 87.5%, 62.5%, 37.5%, 75%, 87.5%., and these three proteins could reduce the tissue damage of Haemophilus accessory pigs to mice. These studies suggest that HxuC, HxuB, and HxuCBA can be used as an effective candidate protein for subunit vaccine.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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