重组SPLUNC1蛋白对人工感染MO的盘羊杂交羊免疫调节作用研究

发布时间：2018-07-07 22:56

本文选题：短腭、肺及鼻咽上皮克隆1蛋白 + 绵羊肺炎支原体　；参考：《石河子大学》2017年硕士论文

【摘要】：目的:本试验首先人工感染绵羊肺炎支原体(MO)得到绵羊肺炎支原体病例,然后用巴什拜羊和盘羊杂交羊重组SPLUNC1蛋白治疗感染MO的盘羊杂交羊,再用ELISA和real-time q PCR的方法,测定IL-5、IL-6、IL-8、IL-9、IL-12和IL-13细胞因子的血清含量和口腔后上腭(咽喉部)上皮组织中SPLUNC1 m RNA的表达水平,以研究重组SPLUNC1蛋白对盘羊杂交羊的免疫调节和治疗效果。方法:(1)SPLUNC1基因的表达和蛋白的纯化:利用课题组已构建好的巴什拜羊和盘羊杂交羊重组GS115/p PIC9K-SPLUNC1巴斯德毕赤酵母阳性克隆菌株,甲醇诱导表达96 h,采用Phenyl sepharose FF树脂进行疏水层析纯化,SDS-PAGE方法检测表达和纯化结果。(2)重组SPLUNC1蛋白对感染MO的盘羊杂交羊相关细胞因子的调节作用:6只巴什拜羊为A组,18只盘羊杂交羊随机分为B、C、D组,全部人工感染MO,每天观察临床症状并记录体温,两周后用ELISA试剂盒检测MO抗体,以验证是否感染MO。从感染第5 d开始,C、D组分别气管注射巴什拜羊和盘羊杂交羊重组SPLUNC1蛋白,150μg/只,每天1次,A、B组气管注射等量生理盐水。分别在第0 d、2 d、5 d、7 d、14 d和21 d采集全部羊的颈静脉血,分离血清,用ELISA方法检测血清中IL-5、IL-6、IL-8、IL-9、IL-12和IL-13的浓度。(3)重组SPLUNC1蛋白对感染MO的盘羊杂交羊的内源SPLUNC1 m RNA水平的影响:在感染前(第0 d)和感染后(第5 d)和治疗后(第21 d),刮取口腔后上腭(咽喉部)上皮组织样品,用real time-q PCR方法检测SPLUNC1 m RNA的表达水平。(4)重组SPLUNC1蛋白对感染MO的盘羊杂交羊的治疗作用:对C、D组连续气管注射巴什拜羊和盘羊杂交羊重组SPLUNC1蛋白,至第21 d,每天测量体温,观察临床症状,试验结束后剖检试验羊,观察肺部变化并照相,取肺组织制作病理切片,采用支原体肺炎组织病理学评分系统,对全部试验羊肺组织病理切片进行病理学评分,评价重组SPLUNC1蛋白的治疗效果。结果:(1)甲醇诱导表达重组GS115/p PIC9K-SPLUNC1巴斯德毕赤酵母阳性克隆株,收集96 h上清液,SDS-PAGE电泳显示有目的条带。重组SPLUNC1蛋白纯化后,SDS-PAGE电泳显示出巴什拜羊25.53 k Da和盘羊杂交羊25.96 k Da的单一目的条带。(2)人工感染MO两周后,所有试验羊MO抗体ELISA检测结果均为阳性,说明全部羊感染了MO。各试验组相关细胞因子变化如下:A组IL-5水平在感染后第14~21 d极显著低于B组(P0.01;P0.01),显著低于C、D组(P0.05;P0.05);在第21 d,C、D组极显著低于B组(P0.01)。C、D组IL-6在感染后第14~21 d极显著低于B组(P0.01;P0.01),A组极显著低于B组(P0.01;P0.01)。IL-8水平在第14~21 d,C、D组显著和极显著低于B组(P0.05;P0.01),A组极显著低于B组(P0.01;P0.01)。B组IL-9水平在感染后第7 d极显著高于A、C、D组(P0.01),在第14~21 d C、D组显著和极显著低于B组(P0.05;P0.01),A组极显著低于B组(P0.01;P0.01)。IL-12水平在第7 d,A组显著低于B组(P0.05);14 d时C、D组显著和极显著低于B组(P0.05;P0.01),A组极显著低于C、D组(P0.01);第21 d,C、D组极显著低于B组(P0.01)。IL-13水平在感染后第5 d,A组显著低于B、C、D组(P0.05);在第7 d,B组显著高于A、C、D组(P0.05);在第14~21 d,C、D组极显著低于B组(P0.01;P0.01);在第21 d,A组显著高于C、D组(P0.05),显著低于B组(P0.05)。(3)在感染后,各组试验羊的SPLUNC1 m RNA水平均升高,C、D组在治疗后SPLUNC1 m RNA水平极显著高于B组(P0.01;P0.01);A组SPLUNC1 m RNA水平也极显著高于B组(P0.01)。(4)A组试验羊2~3 d体温一过性增高(40~41℃),之后体温逐步恢复正常,剖检肺组织有出血点;B组体温升高明显(41~42℃),严重咳嗽、腹泻,剖检发现肺表面有结节和肝变;C组经巴什拜羊重组SPLUNC1蛋白治疗后咳嗽、腹泻等症状缓解,剖检发现肺表面有出血点和出血斑;D组注射盘羊杂交羊重组SPLUNC1蛋白后,其临床症状减轻,剖检发现肺部有出血斑。各组试验羊肺组织病理评分结果分别为,A组8.8分,B组18.7分,C组13.2分,D组14分,C、D组显著低于(P0.05)B组,A组极显著低于B组(P0.01)。结论:成功表达了巴什拜羊和盘羊杂交羊重组SPLUNC1基因,采用Phenyl sepharose FF树脂疏水层析纯化出了单一目的条带,目的条带大小分别为25.53k Da和25.96k Da。C、D组经巴什拜羊和盘羊杂交羊重组SPLUNC1蛋白治疗后,血清中IL-5、IL-6、IL-8、IL-9、IL-12及IL-13的浓度与B组相比有明显变化,说明两种重组SPLUNC1蛋白对感染MO的盘羊杂交羊相关细胞因子有调节作用。各组羊SPLUNC1 m RNA水平结果说明,重组SPLUNC1蛋白对感染MO的盘羊杂交羊内源SPLUNC1 m RNA水平有一定促进作用。剖检及病理切片评分结果表明,以上两种重组SPLUNC1蛋白对感染MO的盘羊杂交羊均有一定的治疗作用。
[Abstract]:Objective: in this experiment, we first acquired Mycoplasma sheeppneumoniae (MO) to obtain Mycoplasma sheeppneumoniae, and then recombined SPLUNC1 protein with bash sheep and cross sheep to treat MO sheep cross sheep. Then ELISA and real-time Q PCR were used to determine the serum content of IL-5, IL-6, IL-8, IL-9, IL-12 and cytokines. The expression level of SPLUNC1 m RNA in the epithelial tissue of the posterior palate (pharynx) in order to study the immunoregulation and therapeutic effect of recombinant SPLUNC1 protein on mutton hybrid sheep. Methods: (1) the expression of the SPLUNC1 gene and the purification of the protein: the recombinant GS115/p PIC9K-SPLUNC1 Pasteur Pichia pastoris has been constructed by the project group. The mother positive cloned strain was induced by methanol to express 96 h, purified by Phenyl Sepharose FF resin, and detected the expression and purification results by SDS-PAGE method. (2) the regulation of recombinant SPLUNC1 protein on the related cytokines of sheep cross sheep infected with MO: 6 basbai sheep were A group, and 18 sheep hybrid sheep were randomly divided into B, C, D group, all Artificial infection of MO, observe the clinical symptoms and record the body temperature every day. After two weeks, the ELISA kit is used to detect MO antibodies to verify whether infection MO. starts from infection fifth D, C, and group D, respectively, to intratrachee the recombinant SPLUNC1 protein of bashbai sheep and cross sheep, 150 u g/, 1 times a day, A, and B group, respectively, in zeroth D, 2, 5, 5, respectively, 7 d, 14 d and 21 d collected all the sheep's jugular vein blood, separated the serum, and detected the concentration of IL-5, IL-6, IL-8, IL-9, IL-12 and IL-13 in the serum by ELISA method. (3) the effect of the recombinant SPLUNC1 protein on the level of the internal origin of the sheep with the infection MO. (zeroth) and after infection (fifth) and after treatment (twenty-first), after scraping the mouth Real time-q PCR method was used to detect the expression level of SPLUNC1 m RNA in the epithelial tissue of the palate (pharynx). (4) the therapeutic effect of recombinant SPLUNC1 protein on the cross sheep with MO in the sheep: C, the D group was continuously injected with bashbai sheep and cross sheep to recombine the SPLUNC1 protein, to twenty-first D, the temperature was measured every day, the clinical symptoms were observed, the experimental knot was observed. The changes of the lung were observed and the lung tissues were observed and photographed. The pathological sections of the lung tissue were made. The pathological grade of mycoplasma pneumonia was used to evaluate the pathological sections of the lung tissue and to evaluate the therapeutic effect of the recombinant SPLUNC1 protein. Results: (1) the recombinant GS115/p PIC9K-SPLUNC1 Pasteur was induced by methanol. The positive clones of red yeast were collected from 96 h supernatant and SDS-PAGE electrophoresis showed a purposeful strip. After the recombinant SPLUNC1 protein was purified, SDS-PAGE electrophoresis showed a single target band of 25.53 K Da and 25.96 K Da in Bash sheep and sheep. (2) after two weeks of artificial infection of MO, all tested MO antibody ELISA detection results were all positive, indicating that all the test results were positive The changes of related cytokines in the MO. group were as follows: the level of IL-5 in the A group was significantly lower than the B group (P0.01; P0.01) in the 14~21 d after infection (P0.01; P0.01), and the D group (P0.05; P0.05) was significantly lower than that of the D group. 0.01).IL-8 level in 14~21 D, C, D group was significantly and significantly lower than group B (P0.05; P0.01), and A group was significantly lower than B group (P0.01; P0.01) was significantly higher than that in the post infection group. Group B was significantly lower than group B (P0.05); at 14 d C, D group was significantly and significantly lower than B group (P0.05; P0.01), A group was significantly lower than C, D group (P0.01). (P0.01; P0.01); in twenty-first D, group A was significantly higher than C, D group (P0.05), significantly lower than B group (P0.05). (3) SPLUNC1 m RNA level increased after infection, and the level of the group was significantly higher than that of the group. (4) Hyperthermia (40~41 C), then the body temperature gradually recovered to normal, the pulmonary tissue had hemorrhagic spots, B group temperature increased significantly (41~42 C), severe cough, diarrhea, the pulmonary surface of the lung nodules and liver changes found, C group after the bash sheep recombinant SPLUNC1 protein treatment cough, abdominal diarrhea and other symptoms remission, found that the lung surface bleeding spots and hemorrhagic spots; D group found that bleeding spots and hemorrhagic spots on the lung surface; D group found to have bleeding spots and bleeding spots on the lung surface; group D group found that bleeding spots and spots on the lung surface; group D group found to have bleeding spots and bleeding spots on the lung surface; group D group The clinical symptoms were relieved and the pulmonary hemorrhage spots were found after the recombinant SPLUNC1 protein of sheep cross sheep. The results of histopathology were 8.8, 18.7, 13.2 in group B, 13.2 in group C, 14 in group D, C in group D significantly lower than in group B (P0.05), and in A group was significantly lower than that in B group (P0.01). Conclusion: Bash and pine were successfully expressed. The recombinant SPLUNC1 gene of the hybrid sheep was purified by Phenyl Sepharose FF resin hydrophobic chromatography. The size of the strip was 25.53k Da and 25.96k Da.C, and the D group was treated with the recombinant SPLUNC1 protein of bahbbai sheep and sheep cross sheep. The results showed that two kinds of recombinant SPLUNC1 protein could regulate the related cytokines of sheep cross sheep infected with MO. The results of SPLUNC1 m RNA in each group showed that the recombinant SPLUNC1 protein had a certain effect on the m RNA level of SPLUNC1 in the goat hybrid sheep infected with MO. The results of caesarean section and pathological slice score showed that the above two kinds of recombinant SPLUNC1 eggs Bai has certain therapeutic effects on the goat sheep infected with MO.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.26

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