重组NDV HN基因减毒鸡白痢沙门菌的构建及其特性研究

发布时间：2018-09-03 10:24

【摘要】：新城疫是由新城疫病毒引发的一种急性和高度接触性禽类疾病,对世界养禽业造成了毁灭性的破坏,世界动物卫生组织将其列入必须进行通报的动物疾病之一。近年来,由于传统疫苗对新城疫预防的种种缺陷,研制一种新型基因工程疫苗来预防和控制新城疫势在必行。鸡白痢沙门菌(S.Pullorum)是目前危害养鸡业的重要细菌之一,药物控制该菌易造成生物安全问题,传统疫苗免疫力不高,因此,将减毒沙门菌作为预防细菌疾病的新型活疫苗是当下控制鸡白痢的首选途径。本试验在减毒S.Pullorum C79-13?crp的基础上,进一步缺失asd基因,利用其质粒平衡致死系统构建重组菌株C79-13Δcrp?asd(p YA-HN),并对其生物学及免疫学特性进行研究。1.减毒S.Pullorum C79-13?crp?asd株的构建及其生物学特性本研究以S.Pullorum C79-13?crp作为受体菌、大肠杆菌χ7213(p REΔasd)作为供体菌,将二者进行接合转移,并通过“两步法”筛选asd基因缺失株,PCR鉴定结果及测序结果显示,成功构建S.Pullorum缺失株C79-13?crp?asd。生物学特性结果表明,该菌在DAP阴性环境中存活的能力丧失,同时也失去了利用麦芽糖、葡萄糖、甘露醇和鼠李糖等碳源的能力;其血清型与野生株C79-13一致,生长速度减慢;能够在体外稳定遗传asd缺失基因;雏鸡口服攻毒试验结果显示重组菌株LD50较野生株C79-13升高约3.1×104倍。2.重组NDV HN减毒S.Pullorum的构建及其生物学特性本试验将PCR扩增出的HN基因片段克隆至无抗性原核表达载体p YA3493,得到重组表达质粒p YA-HN,进而将经PCR及酶切鉴定正确的重组质粒导入C79-13?crp?asd中构建重组菌株C79-13Δcrp?asd(p YA-HN)。该重组菌株的生化特性与生长特性与C79-13?crp?asd基本保持一致;NDV HN基因能够在体外稳定遗传;SDS-PAGE结果显示HN基因在重组菌中得到了表达,Western blot结果表明表达蛋白具有良好的免疫原性;重组菌毒力较野生株C79-13升高约3.4×104倍;定植试验结果显示,重组菌对鸡体具有较好的侵袭力。3.重组菌株C79-13Δcrp?asd(p YA-HN)的免疫学特性研究重组菌株口服免疫雏鸡,对其免疫学特性进行研究。结果表明,对鸡只体重的增加与对照组相比差异不显著(P0.05),能显著刺激鸡体免疫器官指数的增加(P0.05);血清HI抗体测定结果显示,HI抗体水平于免疫后21d时达到最大值(log2=5.3);重组菌免疫后第21d免疫血清中沙门菌Ig G抗体水平显著高于对照组(P0.05);在重组菌株免疫鸡肠道冲洗液中检测到较PBS组高水平的分泌型Ig A(log2=3.56);免疫组所分离的淋巴细胞经体外刺激后其SI在免疫第21d达到最大值,即1.698,与PBS组相比差异显著(P0.05);免疫保护试验结果显示,重组菌免疫组可能100%抵抗沙门菌野生株的感染,对NDV F48E8的攻击有60%的保护率。综上所述,重组菌免疫后在一定时期内能够刺激机体产生有效的体液、黏膜和细胞免疫应答反应,且对S.Pullorum及NDV野毒株的感染具有保护效果。
[Abstract]:Newcastle disease (NDV) is an acute and highly contact avian disease caused by Newcastle disease virus (NDV), which has caused devastating damage to the world poultry industry, and has been listed as one of the animal diseases that must be notified by the World Organization of Animal Health (OIE). In recent years, it is imperative to develop a new genetic engineering vaccine to prevent and control Newcastle disease because of the defects of traditional vaccines. Salmonella pullorum (S.Pullorum) is one of the most important bacteria that harm the chicken industry at present. Drug control of Salmonella pullorum is easy to cause biosafety problems, and the immunity of traditional vaccine is not high. Attenuated Salmonella as a new live vaccine to prevent bacterial disease is the first choice to control chicken pullorum. The recombinant strain C79-13 螖 crp?asd (p YA-HN) was constructed on the basis of attenuated S.Pullorum C79-13?crp and further deletion of asd gene. The biological and immunological characteristics of C79-13 螖 crp?asd (p YA-HN) were studied. Construction and Biological characteristics of attenuated S.Pullorum C79-13?crp?asd strain. In this study, S.Pullorum C79-13?crp was used as receptor strain and 蠂 7213 (p RE 螖 asd (Escherichia coli) as donor strain, and the two strains were conjugated and transferred. The results of identification and sequencing of asd gene deletion strain by "two-step method" showed that the S.Pullorum deletion strain C79-13 was successfully constructed. The biological characteristics showed that the strain lost its ability to survive in DAP negative environment and lost the ability to utilize carbon sources such as maltose, glucose, mannitol and rhamnose, and its serotype was consistent with that of wild strain C79-13, and its growth rate slowed down. The results showed that the LD50 of the recombinant strain was 3.1 脳 10 ~ 4 times higher than that of the wild strain C79-13. Construction and Biological characteristics of attenuated S.Pullorum of Recombinant NDV HN the HN gene fragment amplified by PCR was cloned into the non-resistant prokaryotic expression vector p YA3493, to obtain the recombinant expression plasmid p YA-HN, which was identified correctly by PCR and restriction endonuclease digestion. The recombinant strain C79-13 螖 crp?asd (p YA-HN) was constructed by introducing the plasmid into C79-13?crp?asd. The biochemical and growth characteristics of the recombinant strain were basically consistent with that of C79-13?crp?asd. The results of SDS-PAGE showed that the HN gene was expressed in the recombinant strain. The results showed that the expressed protein had good immunogenicity. The virulence of the recombinant strain was about 3.4 脳 104 times higher than that of the wild strain C79-13, and the results of colonization test showed that the recombinant strain had a better invasiveness to chicken body. Immunological characteristics of Recombinant strain C79-13 螖 crp?asd (p YA-HN) the immunological characteristics of recombinant strain C79-13 螖 crp?asd (p YA-HN) were studied. The results show that The increase in body weight of chickens was not significantly different from that of the control group (P0.05), but it could significantly stimulate the increase of body immune organ index (P0.05), the level of serum HI antibody reached the maximum value (log2=5.3) at 21 days after immunization, and the recombinant bacteria were immunized. The antibody level of Salmonella Ig G in serum was significantly higher than that in the control group on the 21st day after immunization (P0.05), the secretory Ig A (log2=3.56 was detected in the intestinal lavage fluid of the recombinant strain immunized chicken, and the lymphocytes isolated from the immunized group were stimulated in vitro. The SI reached the maximum value on the 21st day after immunization. The results of immunoprotection test showed that 100% of the recombinant bacteria immunized group could resist the infection of Salmonella wild-strain and the protective rate of NDV F48E8 was 60%. To sum up, the recombinant bacteria can stimulate the body to produce effective humoral, mucosal and cellular immune responses in a certain period after immunization, and have protective effect on the infection of S.Pullorum and NDV wild strains.
【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】：S852.6

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