【作者】 卫广森；

【导师】 陆承平； 陈溥言；

【作者基本信息】 南京农业大学 ， 兽医， 2004， 博士

【摘要】 新生仔猪大肠杆菌性腹泻（Colibacillus diarrhea of newborn piglet）是由产肠毒素性大肠埃希氏菌（Enterotoxigenic E. coli, ETEC）引起的以1～7日龄新生仔猪下痢为特征的一种急性致死性多发性传染病。本病在世界范围内广泛流行，是影响仔猪成活率的主要因素之一。随着疫苗的研发与应用，我国新生仔猪大肠杆菌性腹泻得到了一定程度的控制，但疫苗在免疫原性、安全性等方面仍不理想。因此，研制高效、安全的新型多价基因工程疫苗迫在眉睫。 本研究利用分子生物学技术将ST₁基因与K₈₈ac和LT_B基因融合在一起，研制大肠杆菌三价基因工程灭活疫苗，以期达到增加疫苗的安全性，提高疫苗免疫效果的目的，解决新生仔猪大肠杆菌性腹泻免疫预防这一难题。 （1） 根据已发表的K₈₈ac、ST₁和LT_B基因序列，分别设计并合成一对K₈₈ac引物、三对ST₁引物和一对LT_B引物，利用PCR技术，从大肠杆菌C83902质粒中扩增出K₈₈ac基因、ST₁突变基因和LT_B基因。将质粒DNA和扩增的DNA片段进行分离、纯化、限制性内切酶酶切、T4 DNA连接酶连接，获得重组质粒pXK88acS73LT5并转化BL21（DE3）宿主菌，构建了含K₈₈ac-ST₁-LT_B融合基因表达载体的重组菌株BL21（DE3）（pXK88acST3LT5）。经酶切鉴定和DNA序列分析证实，构建的重组质粒pXK88acST3LT5中含有K₈₈ac-ST₁-LT_B融合基因，且基因序列和阅读框架均正确。将重组菌株BL21（DE3）（pXK88acST3LT5）按1％的量接种到含有Kan(50μg·mL^-1)的LB液体培养基中，37℃振荡培养2h，然后用1mmol·L^-1IPTG诱导4h，融合蛋白K₈₈ac-ST₁-LT_B获得高效表达。经ELISA检测，重组菌株表达的K₈₈ac-ST₁-LT_B融合蛋白能够被ST₁单抗、LT_B和K₈₈ac抗体识别。将重组菌株BL21（DE3）（pXK88acST3LT5）培养物上清及菌体裂解物，分别进行了乳鼠灌胃试验，结果均为阴性（C／C≤0.083），这表明该融合蛋白K₈₈ac-ST₁-LT_B已丧失天然ST₁肠毒素的活性。用该工程菌株制备的包涵体粗提物和工程菌灭活疫苗免疫小鼠，再用C83902大肠杆菌强毒菌株攻毒均获得了较好的保护，试新生仔猪大肠杆菌性腹泻际ac一STI一LTI，二价基因「程灭活疫苗的研究与应用 里理里旦巴月里巴里里里里里里里里验结果表明，肠ac一sT一LT。融合蛋白能够诱发小鼠产生抗体，该抗体具有中和天然ST:肠毒素毒性的作用，证实了构建的重组菌株可以作为预防新生仔猪大肠杆菌性腹泻基因工程疫苗的候选菌株。 （2）对构建的表达K::aC一ST!一LT。融合蛋白的重组菌株BL21（DE3）（pXK88aeST3LTS）进行了染色特性、培养特性、生化特性、质粒稳定性、免疫原性、保存条件等实验室试验。结果1一10代菌种染色特性、培养特性、生化特性均符合大肠杆菌特性的要求;在无选择压力（Kan一）T BL21（DE3）（pXK88aeST3LTS）菌株连续培养20代，统计质粒丢失率，其重组质粒保留率为1 00%，表明重组质粒pXK88aeST3LTS在受体菌BL21（DE3）中能较稳定传代:重组菌株BL21（DE3）（pXK88aeST3LTS）经诱导后的培养物，用SDS-聚丙烯酞胺凝胶电泳和薄层凝胶扫描分析，结果表明融合蛋白占菌体总蛋白的相对含量能稳定在73%以上;分另，J用BL21（DE3）（pXK88aeST3LTS）菌株第1代和第10代基础种子的培养物制备的疫苗免疫小鼠后，用1 MLD（5/10吕CFU）强毒菌液攻击，其保护率为90.6，表明1一10代基础种子均可作为疫苗生产用菌株，其免疫原性稳定。因此，基础种子代次暂定为1一10代;菌种保存试验确定，甘油保存菌种在一20℃保存期为2年，冻干保存菌种在一20℃保存期暂定为3年;最小免疫剂量测定结果表明小鼠的最小免疫剂量每只为0.lmL，妊振母猪的最小免疫剂量每头为2.smL;制备的5批实验室产品均安全有效;试验中本疫苗保存18个月，仍具有良好的效力，保护率达8既以上，为了保证疫苗质量，并考虑疫苗在保存和应用中的各种不确定因素，将本疫苗的保存期暂定为12个月。为了进一步验证疫苗的安全性和有效性，在不同地区进行T田间试验，免疫妊娠母猪6 50头，产仔7 1 56头，平均保护率为98.2406，试验证明本疚苗安全性良好，新生仔猪通过吮吸母乳获得被动免疫可有效地预防新生仔猪大肠性腹泻的发生。 （3）以实验室工艺为基础，进行了工业化生产工艺的研究。用发酵罐培养，改良LB培养基培养的菌数与普通肉汤培养基的菌数基本一致，明显高于LB培养基的菌数，根据BL21（DE3）（pXK88aeST3LTS）菌株培养的稳定性和降低生产成本的要求，故改良LB培养基为该菌株的最佳培养基;经诱导剂筛选试验分析，100 mmol·L一，乳糖组诱导效果略低于1 mmol·L一，IpTG组，但优于10 mmol·L一，乳糖组，1 mmol·L一，乳糖组诱导效果最差，在工业化生产上为了降低生产成本，故选择乳糖作为诱导剂;摘要aL21（nE3）（pxK88acsT3LTS）菌株添加终浓度为100 mm。l·L一，乳糖进行诱导后，目的蛋白于Zh呈现表达，随着诱导时间的延长，总菌数和目的蛋白表达总数均相应增加，5一6h趋于稳定，确定了乳糖诱导的浓度为10Omm。l·L一，，诱导时间不低于6h;通气培养条件试验表明，BLzl（。Es）（pxKssacST3LTs）菌株以2、10，mL发酵罐通气培养，在500L·min一?更多还原

【Abstract】 Colibacillus diarrhea of newborn piglet is a kind of the most common infectious disease.which was caused by enterotoxigenic E.coli (ETEC) and characterized by ’"yellow scour" of the 1 to 7-day-old newborn piglet and high morbidity and mortality. With the development and application of vaccine. colibacillus diarrhea of newborn piglet has been controlled to a certain extent. but the innocuity and the immunogenicity of the vaccine are still unsatisfied so that it is exigent to develop a new multivalence gene engineering inactivated vaccine which has high innocuity and immunogenicity.In this study. the genes of ST1 K88ac and LTB were linked and the trivalent gene engineering vaccine K88ac-ST1-LTB was constructed successfully with molecular bio-technology. The purpose of this study was to improve the innocuity and the immunity efficacy of the vaccine and promote prevention level against the colibacilius diarrhea of newborn piglet.(1)According to the published DNA sequence of K88ac.ST and LTb, one pair of K88ac primer, three pairs of ST1 primers and one pair of LTb primer had been designed and synthesized .With the technology of PCR. K88ac gene. ST1 mutant gene and LTb gene were amplified respectively. After isolating . purifying, restriction endonuclease-digesting and linking with T4 DNA Ligase .the recombinant plasmid pXK88acST3LT5 was obtained and transformed into BL21 ( DE3) thus the recombinant strain BL21 C DE3 )(pXK88acST3LT5) expressing fusion protein K88ac-ST1-LTB was constructed . It was proved that the recombinant plasmid pXK88acST3LT5 contained the fusion gene K88ac-ST1-LTB which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis. The recombinant strain BL21 (DE3 )( pXK88acST3LT5) was inoculated into LB medium at the ratio of 1%, and was cultured for 2 hours at 37 . then was induced bv 1mmol L-1 1PTG for 4 h, finally the fusionprotein K88ac-ST1-LTB was expressed at high level. The expressed protein can be recognized by MAb of ST1, antibodies of LTB and K88ac with ELISA method. With the supernatant and lysate of the recombinant strain BL21 (DE3) (pXK88acST3LT5) respectively, the test of pouring stomach of sucking mice was done and the result was negative (G/C 0.083) ,which indicated that the fusion protein lost the toxicity of ST1.The mice had been protected which were immunized with inclusion body or the inactivated vaccine and then challenged with C83902 virulent strain. The result showed that the fusion protein K88ac-ST1-LTB can stimulate the mice to produce antibody which has the function of neutralizing the natural STi toxin. Therefore, the recombinant strain could be used as a candidate strain of gene engineering inactivated vaccine against colibacillus diarrhea of newborn piglet.(2)The recombinant strain BL21 (DE3) (pXK88acST3LT5) was tested for the properties of bacterial staining, culturing, plasmid stability, preservation condition and so on in laboratory. The properties of bacterial staining, culturing, biochemistry of the bacterium seeds from the first to the tenth generation were all in accord with E.coli; Under the unselected pressure (Kan") , the recombinant strain BL21 (DE3) (pXK88acST3LT5) can be cultured for twenty generations continually and the reserved rate of recombinant plasmid was 100%.The result indicated that the recombinant plasmid pXK88acST3LT5 can be transmitted stably in BL21 (DE3) .The induced culture of the recombinant strain BL21 (DE3) (pXK88acST3LT5) was analyzed with SDS-PAGE and thin-layer gel scan, the result showed that the relative content of fusion protein was above 73% of total protein; The mice immunized respectively with the culture of the first and the tenth basic seeds were challenged with 1 MLD (5 108 CFU) virulent bacterium liquid, and the protected rate was 90%, which indicated all of the seeds from the first to the tenth generation can be used to produce vaccine. So they can be chosen as basic seeds .By use of kept term detecting test, the term of seed kept in glycerin at -20 癈 was two years,while it更多还原