本文選題：短腭、肺及鼻咽上皮克隆1蛋白 + 綿羊肺炎支原體　； 參考：《石河子大學》2017年碩士論文

【摘要】：目的:本試驗首先人工感染綿羊肺炎支原體(MO)得到綿羊肺炎支原體病例,然后用巴什拜羊和盤羊雜交羊重組SPLUNC1蛋白治療感染MO的盤羊雜交羊,再用ELISA和real-time q PCR的方法,測定IL-5、IL-6、IL-8、IL-9、IL-12和IL-13細胞因子的血清含量和口腔后上腭(咽喉部)上皮組織中SPLUNC1 m RNA的表達水平,以研究重組SPLUNC1蛋白對盤羊雜交羊的免疫調(diào)節(jié)和治療效果。方法:(1)SPLUNC1基因的表達和蛋白的純化:利用課題組已構建好的巴什拜羊和盤羊雜交羊重組GS115/p PIC9K-SPLUNC1巴斯德畢赤酵母陽性克隆菌株,甲醇誘導表達96 h,采用Phenyl sepharose FF樹脂進行疏水層析純化,SDS-PAGE方法檢測表達和純化結果。(2)重組SPLUNC1蛋白對感染MO的盤羊雜交羊相關細胞因子的調(diào)節(jié)作用:6只巴什拜羊為A組,18只盤羊雜交羊隨機分為B、C、D組,全部人工感染MO,每天觀察臨床癥狀并記錄體溫,兩周后用ELISA試劑盒檢測MO抗體,以驗證是否感染MO。從感染第5 d開始,C、D組分別氣管注射巴什拜羊和盤羊雜交羊重組SPLUNC1蛋白,150μg/只,每天1次,A、B組氣管注射等量生理鹽水。分別在第0 d、2 d、5 d、7 d、14 d和21 d采集全部羊的頸靜脈血,分離血清,用ELISA方法檢測血清中IL-5、IL-6、IL-8、IL-9、IL-12和IL-13的濃度。(3)重組SPLUNC1蛋白對感染MO的盤羊雜交羊的內(nèi)源SPLUNC1 m RNA水平的影響:在感染前(第0 d)和感染后(第5 d)和治療后(第21 d),刮取口腔后上腭(咽喉部)上皮組織樣品,用real time-q PCR方法檢測SPLUNC1 m RNA的表達水平。(4)重組SPLUNC1蛋白對感染MO的盤羊雜交羊的治療作用:對C、D組連續(xù)氣管注射巴什拜羊和盤羊雜交羊重組SPLUNC1蛋白,至第21 d,每天測量體溫,觀察臨床癥狀,試驗結束后剖檢試驗羊,觀察肺部變化并照相,取肺組織制作病理切片,采用支原體肺炎組織病理學評分系統(tǒng),對全部試驗羊肺組織病理切片進行病理學評分,評價重組SPLUNC1蛋白的治療效果。結果:(1)甲醇誘導表達重組GS115/p PIC9K-SPLUNC1巴斯德畢赤酵母陽性克隆株,收集96 h上清液,SDS-PAGE電泳顯示有目的條帶。重組SPLUNC1蛋白純化后,SDS-PAGE電泳顯示出巴什拜羊25.53 k Da和盤羊雜交羊25.96 k Da的單一目的條帶。(2)人工感染MO兩周后,所有試驗羊MO抗體ELISA檢測結果均為陽性,說明全部羊感染了MO。各試驗組相關細胞因子變化如下:A組IL-5水平在感染后第14~21 d極顯著低于B組(P0.01;P0.01),顯著低于C、D組(P0.05;P0.05);在第21 d,C、D組極顯著低于B組(P0.01)。C、D組IL-6在感染后第14~21 d極顯著低于B組(P0.01;P0.01),A組極顯著低于B組(P0.01;P0.01)。IL-8水平在第14~21 d,C、D組顯著和極顯著低于B組(P0.05;P0.01),A組極顯著低于B組(P0.01;P0.01)。B組IL-9水平在感染后第7 d極顯著高于A、C、D組(P0.01),在第14~21 d C、D組顯著和極顯著低于B組(P0.05;P0.01),A組極顯著低于B組(P0.01;P0.01)。IL-12水平在第7 d,A組顯著低于B組(P0.05);14 d時C、D組顯著和極顯著低于B組(P0.05;P0.01),A組極顯著低于C、D組(P0.01);第21 d,C、D組極顯著低于B組(P0.01)。IL-13水平在感染后第5 d,A組顯著低于B、C、D組(P0.05);在第7 d,B組顯著高于A、C、D組(P0.05);在第14~21 d,C、D組極顯著低于B組(P0.01;P0.01);在第21 d,A組顯著高于C、D組(P0.05),顯著低于B組(P0.05)。(3)在感染后,各組試驗羊的SPLUNC1 m RNA水平均升高,C、D組在治療后SPLUNC1 m RNA水平極顯著高于B組(P0.01;P0.01);A組SPLUNC1 m RNA水平也極顯著高于B組(P0.01)。(4)A組試驗羊2~3 d體溫一過性增高(40~41℃),之后體溫逐步恢復正常,剖檢肺組織有出血點;B組體溫升高明顯(41~42℃),嚴重咳嗽、腹瀉,剖檢發(fā)現(xiàn)肺表面有結節(jié)和肝變;C組經(jīng)巴什拜羊重組SPLUNC1蛋白治療后咳嗽、腹瀉等癥狀緩解,剖檢發(fā)現(xiàn)肺表面有出血點和出血斑;D組注射盤羊雜交羊重組SPLUNC1蛋白后,其臨床癥狀減輕,剖檢發(fā)現(xiàn)肺部有出血斑。各組試驗羊肺組織病理評分結果分別為,A組8.8分,B組18.7分,C組13.2分,D組14分,C、D組顯著低于(P0.05)B組,A組極顯著低于B組(P0.01)。結論:成功表達了巴什拜羊和盤羊雜交羊重組SPLUNC1基因,采用Phenyl sepharose FF樹脂疏水層析純化出了單一目的條帶,目的條帶大小分別為25.53k Da和25.96k Da。C、D組經(jīng)巴什拜羊和盤羊雜交羊重組SPLUNC1蛋白治療后,血清中IL-5、IL-6、IL-8、IL-9、IL-12及IL-13的濃度與B組相比有明顯變化,說明兩種重組SPLUNC1蛋白對感染MO的盤羊雜交羊相關細胞因子有調(diào)節(jié)作用。各組羊SPLUNC1 m RNA水平結果說明,重組SPLUNC1蛋白對感染MO的盤羊雜交羊內(nèi)源SPLUNC1 m RNA水平有一定促進作用。剖檢及病理切片評分結果表明,以上兩種重組SPLUNC1蛋白對感染MO的盤羊雜交羊均有一定的治療作用。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.26

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