减毒猪霍乱沙门菌△crp△cya△asd C78-1平衡致死系统及其PRRSV ORF5-ORF6重组菌株的构建

发布时间：2018-05-03 05:32

本文选题：自杀性质粒 + asd平衡致死系统　；参考：《河南科技大学》2015年硕士论文

【摘要】：猪霍乱沙门菌是造成仔猪副伤寒和人类食物中毒的重要人畜共患病病原。通过基因工程缺失致弱得到的沙门菌突变株不仅可以作为高效的弱毒疫苗,还可以进一步引入asd无抗性平衡致死系统将其开发为能携带多种外源保护性抗原的活疫苗载体。因此,筛选合适的减毒菌株构建高效活疫苗载体一直是疫苗学研究的热点之一。本试验首先运用自杀性质粒介导的两步法同源重组技术,在猪霍乱沙门菌单缺失株?crpC78-1的基础上先缺失cya基因,构建了毒力更弱且保留良好免疫原性的双缺失株?crp?cyaC78-1,然后再缺失asd基因将其开发为平衡致死系统。最后以此为载体携带PRRSV ORF5和ORF6融合基因,为开发预防仔猪副伤寒和PRRS的二联疫苗奠定基础。主要研究内容包括:1.猪霍乱沙门菌双缺失株?crp?cyaC78-1的筛选与鉴定以猪霍乱沙门菌单缺失株?crp C78-1为模板,扩增cya基因的上、下臂片段cya1和cya2,然后将二者克隆至中间转移载体pBluescriptSK(+),再将串联的cya1+cya2片段插入自杀性质粒p RE112,最后将重组自杀性质粒pRE?cya导入宿主大肠杆菌χ7213。以χ7213(pRE?cya)为供体菌,?crpC78-1为受体菌,运用自杀性质粒介导的两步法同源重组技术筛选猪霍乱沙门菌双缺失株?crp?cya C78-1。2.猪霍乱沙门菌双缺失株?crp?cyaC78-1的生物学特性检测对双缺失株?crp?cya C78-1的生化和生长特性、缺失基因的稳定性、对小鼠的毒力以及免疫保护等生物学特性进行检测,并将其与?crpC78-1、?cyaC78-1以及疫苗株C500进行比较。研究结果显示,?crp?cya C78-1利用碳源的能力与?cyaC78-1和?crpC78-1完全相同,保留了利用葡萄糖的能力,降低了利用半乳糖的能力,完全丧失了利用麦芽糖、鼠李糖、木糖等碳源的能力;其平均世代间隔为48.2min;其口服LD50是?crp C78-1的204倍,是疫苗株C500的322倍;用其口服免疫小鼠109CFU,能对野毒株10LD50CFU的攻击提供100%的保护。3.减毒猪霍乱沙门菌ΔcrpΔcyaΔasdC78-1平衡致死系统的的构建与鉴定以携带重组自杀性质粒pREΔasd的大肠杆菌χ7213为供体菌,减毒猪霍乱沙门菌双缺失株?crp?cyaC78-1为受体菌,利用自杀性质粒介导的两步法同源重组技术筛选?crp?cya?asd C78-1三缺失株。PCR及测序结果表明?crp?cya?asdC78-1三缺失株构建成功。进而本试验将含asd+基因的质粒pYA3493成功地电转入该三缺失株,构建了平衡致死系统?crp?cya?asdC78-1II(p YA3493),该菌株能够在不含DAP的普通培养基上正常生长。4.重组减毒猪霍乱沙门菌ΔcrpΔcyaΔasdC78-1(p YA-ORF5-ORF6)的构建与鉴定采用RT-PCR扩增PRRSV的ORF5和ORF6基因,并将其先后插入原核表达载体p YA3493,经PCR、酶切及测序鉴定正确之后,将重组载体p YA-ORF5-ORF6电转入减毒猪霍乱沙门菌三缺失株?crp?cya?asd C78-1。PCR及双酶切鉴定结果表明重组减毒猪霍乱沙门菌?crp?cya?asd C78-1(pYA-ORF5-ORF6)构建成功。将该重组菌在LB固体培养基中连续转接60代,每十代进行一次PCR鉴定,均能扩增出预期的目的条带,表明ORF5-ORF6融合基因能稳定遗传于该重组菌。
[Abstract]:Salmonella cholerae is an important zoonotic pathogen causing piglet paratyphoid and human food poisoning. The Salmonella mutant strain obtained by genetic engineering deficiency can not only be used as an efficient weak virus vaccine, but also can be further introduced into the ASD non resistant balanced lethal system to develop it into a variety of exogenous protective antigens. Therefore, it is one of the hotspots in the study of vaccine study to screen the appropriate strain of the attenuated strain to construct high efficient live vaccine. First, the two step homologous recombination technology mediated by suicidal particles is used to construct a less virulence and good retention on the basis of the deletion of the CyA gene on the basis of the single deletion crpC78-1 of the pig cholera cholera Salmonella. The double deletion strain of immunogenicity, CRP? CyaC78-1, and then the deletion of the ASD gene, is developed into a balanced lethal system. Finally, it is used as a carrier to carry the PRRSV ORF5 and ORF6 fusion gene, which lays the foundation for the development of the two joint vaccine to prevent piglet paratyphoid and PRRS. The main contents include: 1. sieves of the double deletion strain of the swine cholera Salmonella, CRP? CyaC78-1 CRP C78-1 was selected and identified as a template for the single deletion of CRP C78-1 of Salmonella cholerae. The upper arm fragments of the CyA gene were amplified by cya1 and cya2, then the intermediate transfer vector pBluescriptSK (+) was cloned and the series cya1+cya2 fragments were inserted into the suicidal P RE112, and the recombinant suicidal pRE? CyA was introduced into the host Escherichia coli x 7213.. Using chi 7213 (pRE? CyA) as donor bacteria and crpC78-1 as receptor bacteria, the biological characteristics of CRP? CyA C78-1.2. swine cholera Salmonella double deletion strain, CRP? CyaC78-1, and the biochemical and growth characteristics of the double missing strains, CRP? CyA C78-1, were screened by the two step homologous recombination technique mediated by suicidal particles. The biochemical and growth characteristics of the double missing strains, CRP? CyA C78-1, were detected. Stability, test the biological characteristics of mice's toxicity and immune protection, and compare it with crpC78-1, cyaC78-1 and vaccine strain C500. The results show that the ability of CRP? CyA C78-1 to use carbon source is exactly the same as cyaC78-1 and crpC78-1, and the ability to use glucose is retained and the ability to use galactose is reduced. The ability to use maltose, rhamnose, xylose and other carbon sources was completely lost; its average generation interval was 48.2min, and its oral LD50 was 204 times of that of CRP C78-1 and 322 times of the vaccine strain C500; it could provide 100% of the 10LD50CFU attack of the wild strain of 10LD50CFU by oral immunization with its oral 109CFU, which could protect the delta virulent Salmonella cholera delta CyA delta CyA Delta asdC78-1 equilibrium. The construction and identification of the dead system were carried out by the Escherichia coli x 7213 carrying the recombinant suicidal particle pRE delta ASD, the double deletion strain of the Salmonella cholera strain and the CRP? CyaC78-1 as the receptor bacteria, using the suicidal particle mediated two step homologous recombination technology, and the.PCR and sequencing results of the CRP? CyA? ASD C78-1 three deletion and sequencing results showed CRP? CyA asdC78-1 Three the deletion strain was successfully constructed. Then the plasmid pYA3493 containing the asd+ gene was successfully transferred into the three deletion strain, and a balanced lethal system was constructed, CRP? CyA? AsdC78-1II (P YA3493). The strain could normally grow on the normal medium without DAP, which could normally grow on the.4. recombinant Salmonella cholera delta CRP delta CyA Delta asdC78-1. The ORF5 and ORF6 genes were constructed and identified by RT-PCR amplification of PRRSV, and then inserted into the prokaryotic expression vector p YA3493. After PCR, enzyme digestion and sequencing identification, the recombinant vector p YA-ORF5-ORF6 was transferred to the three missing strains of the Salmonella cholera, CRP? CyA, ASD and double enzyme cutting identification results showed that the recombinant swine cholera cholera Salmonella was reorganized. The bacteria? CRP? CyA? ASD C78-1 (pYA-ORF5-ORF6) was constructed successfully. The recombinant strain was successively transferred to 60 generations in the solid medium of LB, and every ten generation PCR identification could amplify the expected target band, indicating that the ORF5-ORF6 fusion gene could be inherited from the recombinant bacteria.

【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【参考文献】