Cloning, Expression and Antigenicity of Omp H Gene of Pasteurella multocida
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摘要: 为了对禽多杀性巴氏杆菌（Pm）外膜蛋白H（Omp H）的免疫学活性进行鉴定，本研究根据已发表的多杀性巴氏杆菌X-73株（U50907.1）设计了两对引物，用PCR方法扩增了禽多杀性巴氏杆菌CVCC44801株的Omp H，扩增片段为1 348 bp（ORF为1 056 bp）。扩增的Omp H与GenBank中登录号为AE004439.1（Pm70株）、EF635422.1（C44-1株）、EU016232.1（P52株）、JQ082510.1（XJ121株）、U50907.1（X-73株）、U52200.1（P-1059株）的序列比对分析结果表明，其核苷酸同源性范围在80.2%~98.4%，氨基酸同源性范围在79.6%~98.5%。扩增了去信号肽的Omp H基因，构建原核重组表达质粒pET-Omp H，转化BL21并诱导表达，SDS-PAGE结果表明，表达蛋白约为56 ku，与预期分子量的大小相符；蛋白免疫印迹试验结果表明，该蛋白具有良好的免疫学活性，为建立禽Pm血清学检测方法以及禽多杀性巴氏杆菌外膜蛋白疫苗的研究奠定了基础。Abstract: The immunological activity of the outer membrane protein H (Omp H) of Pasteurella multocida (Pm) was identified. Based on the published data on P. multocida strain X-73 (U50907.1), two pairs of primers were used to amplify the Omp H of P. multocida strain CVCC 44801 by PCR.The amplified fragment was determined to be 1 348 bp (ORF at 1 056 bp). The sequence alignment on Omp H of Pm70, C44-1, P52, XJ121, X-73 and P-1059 with those registered in GenBank showed a nucleotide homology between 80.2% and 98.4% and an amino acid homology between 79.6% and 98.5%. The recombinant plasmid pET-Omp H was constructed and transformed into BL21. The expressed protein was about 56 ku, which was close to the expected molecular weight. The western blot also confirmed its immunological activity. The results would provide the information for the establishment of a viable serological detection method and an applicable Omp vaccine against P. multocida.
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Key words:
- Pasteurella multocida /
- Omp H gene /
- prokaryotic expression
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图 1 PCR产物电泳
注：1为HF1和HR1引物的PCR扩增产物；2为HF2和HR2引物的PCR扩增产物；M为DL 2 000 DNA Marker。
Figure 1. Electrophoresis pattern of PCR products
图 3 pET-OmpH重组质粒的酶切鉴定
注：1~3为重组质粒pET-Omp H双酶切；M1为DL 2 000 DNA Marker；M2为DL 15 000 DNA Marker。
Figure 3. Identification of recombinant plasmid pET-OmpH by restricted enzyme digestion
图 4 Omp H基因原核表达SDS-PAGE电泳结果
注：1为Omp H基因表达蛋白的SDS-PAGE结果；2为空质粒阴性对照；3为空菌的阴性对照；M为蛋白质Marker。
Figure 4. Prokaryotic expression of Omp H gene
图 5 Omp H基因的原核表达western blot检测结果
注：1为空质粒标签蛋白的Western blot；2为重组质粒pET-Omp H标签蛋白的western blot；3为重组质粒pET-Omp H目的蛋白的Western blot；M为蛋白质Marker。
Figure 5. Western blot test results on prokaryotic expression of Omp H gene
表 1 引物信息
Table 1. Information on primer
基因 引物名称 引物序列(5′→3′) 扩增片段/bp Length HF1 GACAAGAAAACGGAGCTTTGC 1348 HR1 CCATTCCTTGCAACATATTG OmpH HF2 AGCGGATCCGCAACAGTTTACAATCAAGAC 996 HR2 CGCCTCGAGTTAGAAGTGTACGCGTAAAC 
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[1] 刘金华, 甘孟侯.中国禽病学[M].第2版.北京:中国农业出版社, 2016:205-216. [2] CHEVALIER G, DUCLOHIER H, THOMAS D, et al. Purification and characterization of protein H, the major porin of Pasteurella multocida[J]. Journal of Bacteriology, 1993, 175:266-276. doi: 10.1128/jb.175.1.266-276.1993 [3] LUO Y, GLISSON J R, JACKWOOD M W, et al. Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73[J]. Journal of Bacteriology, 1997, 179(24):7856-7864. doi: 10.1128/jb.179.24.7856-7864.1997 [4] CARTER G R. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types[J]. American Journal of Veterinary Research, 1955, 16(60):481-484. http://cn.bing.com/academic/profile?id=61a1c46eae6cc5dbfb6174d403ff5f0d&encoded=0&v=paper_preview&mkt=zh-cn [5] HEDDLESTON K L, GALLAGHER J E, REBERS P A. Fowl Cholera:gel diffusion precipitin test for serotyping Pasteurella multocida from avian species[J]. Avian Disease, 1972, 16(4):925-936. doi: 10.2307/1588773 [6] 程龙飞, 许利娜, 黄瑜, 等.福建及邻近省份禽源多杀性巴氏杆菌的分离鉴定[J].中国动物传染病学报, 2014, 22(4):40-44. http://www.cqvip.com/QK/98248A/201404/662007588.html [7] LEE J, KIM Y B, KWON M. Outer membrane protein H for protective immunity against Pasteurella multocida[J]. Journal of Microbiology, 2007, 45(2):179-184. http://cn.bing.com/academic/profile?id=e0d3e0b9e8936daf317a5eea9afc685c&encoded=0&v=paper_preview&mkt=zh-cn [8] VASFI M M, MITTAL K R. Role of Outer Membrane Protein H (omPH)-and OmpA-specific Monoclonal Antibodies from Hybridoma Tumors in Protection of Mice Against Pasteurella multoeida[J]. Infection lmmunity, 1997, 65(11):4502-4508. [9] STHITMATEE N, NUMEE S, KAWAMOTO E, et al. Protection of chickens from fowl cholera by vaccination with recombinant adhesive protein of Pasteurella multocida[J]. Vaccine, 2008, 26(19):2398-2407. doi: 10.1016/j.vaccine.2008.02.051 [10] 陈红梅, 施少华, 程龙飞, 等.禽多杀性巴氏杆菌C48-1株Omp基因克隆及序列分析[J].福建农业学报, 2008, 23(4):351-354. http://kns.cnki.net/KCMS/detail/detail.aspx?filename=fjnx200804005&dbname=CJFD&dbcode=CJFQ [11] 高明燕, 徐步, 龚建森, 等. 8株禽多杀性巴氏杆菌外膜蛋白H基因序列分析[J].中国畜牧兽医, 2013, 40(12):43-47. doi: 10.3969/j.issn.1671-7236.2013.12.008 [12] 曹素芳, 黄青云.禽多杀性巴氏杆菌C48-1成熟外膜蛋白H重组亚单位疫苗免疫效果的研究[J].中国兽医科学, 2006, 36(6):464-467. https://www.wenkuxiazai.com/word/0df77b5c804d2b160b4ec0e6-1.doc -
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