本文選題：副豬嗜血桿菌 + hxuCBA��； 參考：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：由副豬嗜血桿菌(Haemophlius parasuis, HPS)引起的Glasser's'病已經(jīng)給全球養(yǎng)豬業(yè)造成重大損失。鐵幾乎是所有生物體不可或缺的微量營養(yǎng)元素,它可以作為多種生物過程的電子受體或供體發(fā)揮作用。血紅素是細(xì)菌重要的鐵源之一,由hxuCBA基因簇組成的hxu血紅素?cái)z取系統(tǒng)是副豬嗜血桿菌獲取鐵源的途徑之一。本文通過對(duì)組成該系統(tǒng)的三個(gè)基因進(jìn)行克隆表達(dá),并分別對(duì)表達(dá)產(chǎn)物進(jìn)行了生物信息學(xué)分析、免疫原性、免疫保護(hù)性、抗血清殺菌等研究。主要研究結(jié)果如下：1.HPS hxuCBA基因簇的克隆、鑒定及分析針對(duì)HPS hxuC、hxuB、hxuA基因(組成hxu血紅素?cái)z取系統(tǒng)的基因)分別設(shè)計(jì)一對(duì)引物(各基因片段中信號(hào)肽序列已去除),基因片段大小分別為2064bp、 1617bp、2847bp。通過PCR的方法從HPS四川M-3分離株的基因組中擴(kuò)增得到hxuC、hxuB、hxuA基因,分別構(gòu)建pMD19-T-C、pMD19-T-B、pMD19-T-A重組克隆質(zhì)粒,三種質(zhì)粒經(jīng)測序鑒定顯示突變堿基未形成終止密碼子,與HPS SH0165菌株的核苷酸序列一致性分別達(dá)99.22%、99.13%、98.11%,hxuC和hxuB基因序列無堿基缺失,hxuA基因序列有6個(gè)堿基缺失。對(duì)HPS HxuC、HxuB、HxuA氨基酸序列進(jìn)行生物信息學(xué)分析,結(jié)果顯示HxuC、HxuB、HxuA成熟期蛋白分別由688、539、949個(gè)氨基酸組成,蛋白大小分別為78.07kDa、60.72kDa、105.72kDa,等電點(diǎn)分別為9.23、9.34、6.36,均無跨膜區(qū)存在。三種蛋白的二級(jí)結(jié)構(gòu)均由無規(guī)則卷曲(30.81%、30.80%、38.78%)、延伸鏈(19.48%、28.76%、29.08%)、α螺旋(40.55%、28.57%、22.13%)和p轉(zhuǎn)角(9.16%、11.87%、10.01%)組成。HxuC口HxuB屬于外膜蛋白,HxuA屬于胞外蛋白。通過功能位點(diǎn)預(yù)測分析,三種蛋白均含有多個(gè)糖基化、磷酸化位點(diǎn),另外HxuC、HxuB、HxuA分別含有1個(gè)TonB依賴受體蛋白信號(hào)2位點(diǎn)、1個(gè)ATP/GTP結(jié)合位點(diǎn)基序A(P環(huán))、1個(gè)內(nèi)質(zhì)網(wǎng)定位序列�？乖砦环治鲲@示HxuC、HxuB、HxuA蛋白均含有多個(gè)抗原決定簇位點(diǎn),并且分別有30、20、38個(gè)與細(xì)胞毒性T細(xì)胞結(jié)合的抗原表位位點(diǎn)。同時(shí)對(duì)HxuC進(jìn)行同源建模,發(fā)現(xiàn)HxuC蛋白在同種間同源性很高,而在同屬與同科間同源性很低。2. HPS hxuCBA基因簇的表達(dá)及純化利用BamHI和Ncol對(duì)pMD19-T-C、pMD19-T-B、pMD19-T-A和pET39b進(jìn)行雙酶切,16℃進(jìn)行連接,構(gòu)建pET39-C、pET39-B、pET39-A重組表達(dá)質(zhì)粒,將重組表達(dá)質(zhì)粒轉(zhuǎn)入E.coil(BL21)表達(dá)菌內(nèi)進(jìn)行誘導(dǎo)表達(dá)。優(yōu)化蛋白表達(dá)條件的結(jié)果顯示,HxuC經(jīng)37℃,0.2 mmol/L IPT G濃度誘導(dǎo)6 h達(dá)到最大表達(dá)量,HxuB經(jīng)37℃,1.2mmol/L IPTG濃度誘導(dǎo)3 h達(dá)到最大表達(dá)量,HxuA在30℃,IPTG誘導(dǎo)濃度0.8mmol/L誘導(dǎo)2 h表達(dá)量最大。經(jīng)SDS-PAGE顯示三種重組蛋白均可以可溶性和包涵體形式存在,分子量大約為105kDa、87kDa、130 kDa(含載體部分)。然后通過鎳離子親和層析法純化得到目的蛋白,純化的蛋白經(jīng)透析后進(jìn)行western-blot結(jié)果表明三種蛋白均具有良好的反應(yīng)原性。3.HPS HxuC、HxuB、HxuA蛋白的免疫特性研究以昆明小鼠作為實(shí)驗(yàn)動(dòng)物,用自制的HxuC、HxuB、HxuA、HxuCBA (包含HxuC、 HxuB、HxuA)疫苗和HPS M-3全菌滅活苗進(jìn)行皮下注射免疫。一免后第14天進(jìn)行二免。一免和二免分別用弗氏完全佐劑和弗氏不完全佐劑。并分別用佐劑和PBS作為對(duì)照。每次免疫后7天對(duì)小鼠進(jìn)行斷尾收集血清。通過ELISA的方法檢測抗體水平和細(xì)胞因子水平,結(jié)果顯示小鼠抗1HxuC、HxuB、HxuA、HxuCBA、M-3血清的抗體效價(jià)分別達(dá)到1：6400、1：3200、1：1600、1：12800、1：12800；相比于免疫前,細(xì)胞因子IL-2、IL-4、IFN-γ的水平均有所升高,而且一免和二免結(jié)果呈現(xiàn)遞增趨勢。本研究進(jìn)行了抗血清殺菌實(shí)驗(yàn),結(jié)果顯示HxuC、HxuB、HxuA、HxuCBA、M-3、佐劑、PBS組的細(xì)菌存活率分別為28.2%、32.3%、38.6%、35.1%、28.5%、46.9%、48.3%。由此可見,HxuC、HxuB、HxuCBA 對(duì) HPS M-3有明顯的抗菌作用,而HxuA的抗菌作用相對(duì)較弱。以昆明小鼠作為實(shí)驗(yàn)動(dòng)物,測出HPS M-3菌株的半數(shù)致死量LD50,約為1.3×109cfu。隨后進(jìn)行了攻毒保護(hù)試驗(yàn),免疫程序同上。另外設(shè)立全菌滅活免疫組。二免后第7天對(duì)小鼠進(jìn)行腹腔攻毒(5×LD50),攻毒后統(tǒng)計(jì)小鼠發(fā)病死亡狀況。結(jié)果顯示PBS對(duì)照組和佐劑對(duì)照組小鼠全部死亡,HxuC、HxuB、HxuA、 HxuCBA、M-3免疫組保護(hù)率為87.5%、62.5%、37.5%、75.0%、87.5%。另外,這三種蛋白均能減少副豬嗜血桿菌對(duì)小鼠的組織損傷。這些研究表明HxuC、HxuB、 HxuCBA可作為亞單位疫苗的有效候選蛋白。
[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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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