本文選題：自殺性質粒 + asd平衡致死系統(tǒng)　； 參考：《河南科技大學》2015年碩士論文

【摘要】：豬霍亂沙門菌是造成仔豬副傷寒和人類食物中毒的重要人畜共患病病原。通過基因工程缺失致弱得到的沙門菌突變株不僅可以作為高效的弱毒疫苗,還可以進一步引入asd無抗性平衡致死系統(tǒng)將其開發(fā)為能攜帶多種外源保護性抗原的活疫苗載體。因此,篩選合適的減毒菌株構建高效活疫苗載體一直是疫苗學研究的熱點之一。本試驗首先運用自殺性質粒介導的兩步法同源重組技術,在豬霍亂沙門菌單缺失株?crpC78-1的基礎上先缺失cya基因,構建了毒力更弱且保留良好免疫原性的雙缺失株?crp?cyaC78-1,然后再缺失asd基因將其開發(fā)為平衡致死系統(tǒng)。最后以此為載體攜帶PRRSV ORF5和ORF6融合基因,為開發(fā)預防仔豬副傷寒和PRRS的二聯(lián)疫苗奠定基礎。主要研究內容包括:1.豬霍亂沙門菌雙缺失株?crp?cyaC78-1的篩選與鑒定以豬霍亂沙門菌單缺失株?crp C78-1為模板,擴增cya基因的上、下臂片段cya1和cya2,然后將二者克隆至中間轉移載體pBluescriptSK(+),再將串聯(lián)的cya1+cya2片段插入自殺性質粒p RE112,最后將重組自殺性質粒pRE?cya導入宿主大腸桿菌χ7213。以χ7213(pRE?cya)為供體菌,?crpC78-1為受體菌,運用自殺性質粒介導的兩步法同源重組技術篩選豬霍亂沙門菌雙缺失株?crp?cya C78-1。2.豬霍亂沙門菌雙缺失株?crp?cyaC78-1的生物學特性檢測對雙缺失株?crp?cya C78-1的生化和生長特性、缺失基因的穩(wěn)定性、對小鼠的毒力以及免疫保護等生物學特性進行檢測,并將其與?crpC78-1、?cyaC78-1以及疫苗株C500進行比較。研究結果顯示,?crp?cya C78-1利用碳源的能力與?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,完全喪失了利用麥芽糖、鼠李糖、木糖等碳源的能力;其平均世代間隔為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,能對野毒株10LD50CFU的攻擊提供100%的保護。3.減毒豬霍亂沙門菌ΔcrpΔcyaΔasdC78-1平衡致死系統(tǒng)的的構建與鑒定以攜帶重組自殺性質粒pREΔasd的大腸桿菌χ7213為供體菌,減毒豬霍亂沙門菌雙缺失株?crp?cyaC78-1為受體菌,利用自殺性質粒介導的兩步法同源重組技術篩選?crp?cya?asd C78-1三缺失株。PCR及測序結果表明?crp?cya?asdC78-1三缺失株構建成功。進而本試驗將含asd+基因的質粒pYA3493成功地電轉入該三缺失株,構建了平衡致死系統(tǒng)?crp?cya?asdC78-1II(p YA3493),該菌株能夠在不含DAP的普通培養(yǎng)基上正常生長。4.重組減毒豬霍亂沙門菌ΔcrpΔcyaΔasdC78-1(p YA-ORF5-ORF6)的構建與鑒定采用RT-PCR擴增PRRSV的ORF5和ORF6基因,并將其先后插入原核表達載體p YA3493,經PCR、酶切及測序鑒定正確之后,將重組載體p YA-ORF5-ORF6電轉入減毒豬霍亂沙門菌三缺失株?crp?cya?asd C78-1。PCR及雙酶切鑒定結果表明重組減毒豬霍亂沙門菌?crp?cya?asd C78-1(pYA-ORF5-ORF6)構建成功。將該重組菌在LB固體培養(yǎng)基中連續(xù)轉接60代,每十代進行一次PCR鑒定,均能擴增出預期的目的條帶,表明ORF5-ORF6融合基因能穩(wěn)定遺傳于該重組菌。
[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.

【學位授予單位】：河南科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

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