| 摘要: |
| 【目的】克隆猪流产嗜性衣原体青海株主要外膜蛋白（Major outer membrane protein,MOMP)基因，并进行序列分析及原核表达。【方法】根据 GenBank 公布的猪流产嗜性衣原体MOMP基因的核苷酸序列,设计并合成4条特异性引物，用套式PCR方法扩增猪流产嗜性衣原体青海株MOMP基因，将其克隆入pMD18-T载体中，进行测序及序列分析。然后将MOMP基因亚克隆入原核表达载体pGEX4T-1中，在大肠杆菌BL21(DE3)中用IPTG诱导表达，对表达产物进行SDS-PAGE和Western blot检测。【结果】扩增到猪流产嗜性衣原体青海株1 170 bp的MOMP全长基因。序列分析结果表明，该基因与已发表的猪流产嗜性衣原体B11001株和CP/12株核苷酸同源性均为99.7%。SDS-PAGE电泳可检测到分子质量约为66 ku的融合蛋白，主要以包涵体形式存在。Western-blot分析表明，重组蛋白可被猪流产嗜性衣原体抗体识别。【结论】成功克隆了猪流产嗜性衣原体青海株MOMP基因，并进行了原核表达，表达的蛋白具有抗原活性。 |
| 关键词: 猪 流产嗜性衣原体 MOMP蛋白 原核表达 |
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| 基金项目:国家科技基础性工作专项（2008FY210200）；陕西省科技攻关项目（2009K02-01）；西北农林科技大学“青年学术骨干”支持计划项目（E111020901） |
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| Cloning and prokaryotic expression of the major outer membrane protein coding gene of Swine Chlamydophila abortus |
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| Abstract: |
| 【Objective】The outer membrane protein （MOMP）gene of Swine Chlamydophila abortus（CA）Qinghai strain was cloned,analyzed and expressed in E.coli.【Method】According to the published complete nucleotide sequence of CA in GenBank，four primers specific to the full-length of MOMPgene were designed.The MOMP gene fragment amplified by nested PCR was cloned into pMD18-Tvector.The recombinant plasmid was sequenced and MOMP gene was compared with other GeneBank’s CA strains.MOMP gene segment was amplified from recombinant plasmid by PCR,then the MOMPgene was subcloned into vector pGEX4T-1 for prokaryotic expression in BL21(DE3) E.coli induced by IPTG.【Result】The MOMP gene of Qinghai strain was 1 170 bp in full-length.It shared 99.7% nucleotide acid sequences homology with different CA strains(B11001 and CP/12).The recombinant fusion proteins were highly efficiently expressed in E.coli BL21 in the form of inclusion body.The molecular weight of the expressed fusion protein was approximately 65 ku.Western blot showed that the recombinant protein can be recognized by Chlamydophila abortus positive serum.【Conclusion】CA Qinghai strain MOMP gene was cloned and expressed in E.coli successfully and the expressed protein had antigen activity . |
| Key words: swine Chlamydia abortus MOMP prokaryotic expression |
