奶牛乳房炎金黄色葡萄球菌FnBPA D区基因的克隆与表达
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摘要
金黄色葡萄球菌是引起奶牛乳腺炎的一种主要传染性病原菌。本实验运用了分子克隆技术及蛋白表达技术,成功的表达了金黄色葡萄球菌纤维素结合蛋白A(FnBPA)D区的蛋白并对其免疫学活性进行了研究。
本研究根据GenBank上公布的金黄色葡萄球菌FnBPA D区的序列,设计了特异性引物,提取分离自内蒙古地区的金黄色葡萄球菌基因11型和基因3型的基因组DNA后进行PCR,并与T载体连接进行克隆。克隆产物经核酸序列测定后表明已成功克隆了目的基因。测序后分别与金黄色色葡萄球菌NCTC 8325株的FnBPA进行比对发现,基因11型和基因3型与其核苷酸同源性分别为95%和94%。基因3型与基因11型之间核苷酸的同源性为96.5%。
用DNAstar对测序片段进行抗原位点分析,结果显示,基因11型要比基因3型和标准菌株多一个抗原位点,由此决定表达基因11型的FnBPA D区蛋白。将目的片段与表达载体pET-32(a)连接,连接产物转化宿主菌BL21(DE3)后,用IPTG进行诱导,确定最佳诱导时间,大量表达并纯化出了目的蛋白。
表达产物经两次免疫实验动物后,获得较高效价的抗体。用自制血清进行吞噬调理试验,抗金黄色葡萄球菌黏附等一系列试验,证明抗体对金黄色葡萄球菌具有吞噬调理作用,也有抗金黄色葡萄球菌黏附的作用,这将为制备奶牛乳房炎金黄色葡萄球菌黏附素疫苗奠定基础。
The staphylococcus aureus is a kind of main contagion cause germ which causes milk cow mastitis.This study using molecular cloning technique and protein expression technique expresses region D of fibronectin-binding protein A gene of Staphylococcus aureus successfully and research its immunologic competence.
Region D of fibronectin-binding protein A gene (FnBPA gene)of Staphylococcus aureus is amplified with PCR method, on the basis a pair of specific primers designed according to the relevant nucleotide sequence from GeneBank. We use genome DNA of gene type 3 and gene 11 which separated from Inner Mongolia .After PCR, we complete cloning of FnBPA D region. The positive recombinant clones was sequenced and analysed. The results suggested that FnBPA D region gene was cloned successfully. Then we blast the result with relevant nucleotide sequence on GeneBank. The result shows that the autoploidies of gene type 3 and gene 11 with the sequence on GeneBank are 98% and 94%. And the autoploidy of gene type 3 and gene 11 with is 96.5%.
Antigen sites are analysised by the software DNAstar, the result shows gene type 3 has more antigen site than the other two sequences. So we decide to express the gene type 3. The fragment is successfully cloned into pET-32a(+)to construct a recombinant expressed vector pET- FnBPA. The recombinant expressed plasmid is transformed into the host cell E.coli BL21(DE3)and induced by IPTG and then depurated the protein.
After experimental animals are immuned by expression product two times, we obtain high potency antibody. Then carry out regulation and anti-binding of staphylococcus aureus experiments, and the results show that the anbody has the contribution of immunoprotection and anti-binding of staphylococcus aureus. This can play a role in research pathogenesis of staphylococcus aureus and preparing vaccine for mastitis.
本研究根据GenBank上公布的金黄色葡萄球菌FnBPA D区的序列,设计了特异性引物,提取分离自内蒙古地区的金黄色葡萄球菌基因11型和基因3型的基因组DNA后进行PCR,并与T载体连接进行克隆。克隆产物经核酸序列测定后表明已成功克隆了目的基因。测序后分别与金黄色色葡萄球菌NCTC 8325株的FnBPA进行比对发现,基因11型和基因3型与其核苷酸同源性分别为95%和94%。基因3型与基因11型之间核苷酸的同源性为96.5%。
用DNAstar对测序片段进行抗原位点分析,结果显示,基因11型要比基因3型和标准菌株多一个抗原位点,由此决定表达基因11型的FnBPA D区蛋白。将目的片段与表达载体pET-32(a)连接,连接产物转化宿主菌BL21(DE3)后,用IPTG进行诱导,确定最佳诱导时间,大量表达并纯化出了目的蛋白。
表达产物经两次免疫实验动物后,获得较高效价的抗体。用自制血清进行吞噬调理试验,抗金黄色葡萄球菌黏附等一系列试验,证明抗体对金黄色葡萄球菌具有吞噬调理作用,也有抗金黄色葡萄球菌黏附的作用,这将为制备奶牛乳房炎金黄色葡萄球菌黏附素疫苗奠定基础。
The staphylococcus aureus is a kind of main contagion cause germ which causes milk cow mastitis.This study using molecular cloning technique and protein expression technique expresses region D of fibronectin-binding protein A gene of Staphylococcus aureus successfully and research its immunologic competence.
Region D of fibronectin-binding protein A gene (FnBPA gene)of Staphylococcus aureus is amplified with PCR method, on the basis a pair of specific primers designed according to the relevant nucleotide sequence from GeneBank. We use genome DNA of gene type 3 and gene 11 which separated from Inner Mongolia .After PCR, we complete cloning of FnBPA D region. The positive recombinant clones was sequenced and analysed. The results suggested that FnBPA D region gene was cloned successfully. Then we blast the result with relevant nucleotide sequence on GeneBank. The result shows that the autoploidies of gene type 3 and gene 11 with the sequence on GeneBank are 98% and 94%. And the autoploidy of gene type 3 and gene 11 with is 96.5%.
Antigen sites are analysised by the software DNAstar, the result shows gene type 3 has more antigen site than the other two sequences. So we decide to express the gene type 3. The fragment is successfully cloned into pET-32a(+)to construct a recombinant expressed vector pET- FnBPA. The recombinant expressed plasmid is transformed into the host cell E.coli BL21(DE3)and induced by IPTG and then depurated the protein.
After experimental animals are immuned by expression product two times, we obtain high potency antibody. Then carry out regulation and anti-binding of staphylococcus aureus experiments, and the results show that the anbody has the contribution of immunoprotection and anti-binding of staphylococcus aureus. This can play a role in research pathogenesis of staphylococcus aureus and preparing vaccine for mastitis.
引文
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2. 苑方重, 张继东, 钟绣会,等.奶牛乳房炎研究现状[J],动物医学进展. 2003,24(4):44-45
3. Charls N, Dobbins J R.Mastitis losses [J]. J Am Vet Med Assoc,1997, 170:1129-1132.
4. Jose A, Aldo CA, horaco R, et al. Field trials of a vaccine against bovine mastitis: Evaluationinheifers [J]. JDairySci, 1997, 80: 845-853.
5. Simth K.L,et al.Proceedings of the 3rd International Mastitis seminar May 28-June 1995 Tel Aviv Israel s6.3-12
6. Elina Reinoso, GabrielMagnano, Jose Giraudo. Bovine and RabbitModels for the Study of a S taphylococcus aureus AvirulentMutant Strain, RC122 [J]. Can J Vet Res, 2002, 66 (4):285 -288.
7. Talbot Brian G, Lacasse Pierre. Progress in the Development of Mastitis, Vaccines [J]. Livestock Production Science, 2005, 98(1/2):101-113.
8. 聂爱琴,郑明学.奶牛乳腺炎的病因分析[J].科技情报开发与经济,2002,13(3):183-184
9. 李新圃,张礼华.奶牛乳腺炎主要致病菌的药敏试验[J].中国兽医科技,1995,25(9):27-28
10. Naidu A.S,Forsgren r,et al.Comparison between lactoferin and subepothelial matrix Protein binding in Staphylococcus aureus associated with bovine mastitis [J]. Dairy Sci,1991,74:3353
11. Hogan J.S.et al.J.Dairy Sci,1989,72:1547-1556
12. Minion F C. Molecular Pathogenesis of Mycoplasma Animal Respiratory Pathogens [J]. Front Biosci.2002,(7):1410-1422
13. 刘学忠,王捍东.奶牛乳房炎的病因分析[J].中国草食动.2002,22(3):39-41
14. Emanuelson U, Danell B, Philipsson J. Genetic Parameters for Clinical Mastitis, Somatic Cell Counts, and Milk Production Estimated by Multiple-Trait Restricted Maximum Likelihood [J]. J Dairy Sci.1998,(71):467-476
15. Rosenbusch R F, Whitford H W, Rosenbusch R F, Lauerman L H. et al. Mycoplasmosis in Animals: Laboratory Diagnos [M].1994:3-11
16. Maddox J.F et al.J.Dairy Sci,1990,73:Supplcment 1,151
17. Schukken Y.H et al.Vet.Rec,1989,125:393-396
18. 贾玉萍, 万仁忠, 宁召峰, 等. 奶牛乳房炎的病原菌分离鉴定与药物敏感性试验[J], 中国畜牧兽医. 2004,31(10):46-48.
19. 史冬艳,郝永清.奶牛乳房炎金黄色葡萄球菌疫苗研究进展[J]. 中国兽药杂志. 2007,41(2): 46-49.
20. 郝建国,梁淑萍.日本学者对体细胞数与乳房炎关系的论述[J].中国奶牛,2002,1:52-54
21. 李勇,葛秀国.奶牛阴性乳房炎研究进展[J].黄牛杂志,2003,29(3):38-41
22. K larry smith A look at physiological and regulatory scc standards in milk[N/OL].IDF Mastitis Newsletter “udder topics”,September 1996
23. Hoie S 等.一种检测葡萄球菌 DNA 酶抗体的方法[J].国外兽医学-畜禽传染病,1991,11(3):36-37
24. 储明星,师守坊.奶牛体形和体细胞数(乳房炎)之间关系的研究进展[J].畜牧与兽医,1995 27(1):35-37
25. 高星 译.如何控制并保持牛奶中体细胞数不超过 10 万[J].中国乳业,2003,5:14-16
26. h.Dosogne,F.Vangroenweghe et al.Differntial leukocyte count method for bovine low somatic cell count milk [J].Dairy sci,2002,86:828-834
27. 唐双喜.冷冻对奶样中体细胞计数影响的研究[J].北京奶业,1998,1:27-30
28. 甘肃农业大学主编.乳腺炎[M].兽医产科学,1980,353-359
29. Fitzgerald J R,Meaney W J,Hartigani P J. Fine-structure Molecular Epidemiological Analysis of Staphylococcus aureus Recovered from Cows [J]. Epidemiol Infect,1997, 119(5):261-269
30. 邓志爱,李孝权,张健等. 食源性金黄色葡萄球菌致病分离株 RAPD 分子分型研究[J]. 中国卫生检验杂志,2006,16(5):619-620
31. Sutra L,Mendolia C,Rainard P. Encapsulation of Staphylococcus aureus Isolates from Mastitic Milk: Relationship between Capsular Polysaccharide Types 5 and 8 and Colony Morphology in Serum-soft Agar,Clumping Factor,Teichoic Acid,and Protein A[J].J Clin Microbiol,1990,28(3):447-451
32. Albert Guidry,Ali Fattom,Atul Pate. Prevalence of Capsular Serotypes among Staphylococcus aureus Isolates from Cows with Mastitis in the United States [J]. Veterinary Microbiology,1997,59(1):53-58
33. Guidry A,Fattom A,Patel A.Serotyping Scheme for Staphylococcus aureus Isolated from Cows with Mastitis [J].Am J Vet Res,1998,59(12):1537-1539
34. Patti JM, Allen BL, McGavin MJ. MSCRAMM-mediated adherence ofmicroorganisms to host tissues [J]. Annu. Rev. Microbiol., 1994,48: 585-617
35. Zhou HongXiong, Zheng-Ying Li, Han-Ping. An immunogenicity study of a newly fusion protein Cna-FnBP vaccinated against Staphylococcus aureus infections in a mice model: Vaccine.2006,24( 22):4830-4837.
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