Monoclonal antibodies to Escherichia coli-expressed P46 and P65 membranous proteins for specific immunodetection of Mycoplasma hyopneumoniae in lungs of infected pigs
Monoclonal antibodies to Escherichia coli-expressed P46 and P65 membranous proteins for specific immunodetection of Mycoplasma hyopneumoniae in lungs of infected pigs
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DOI：
10.1128/cdli.10.3.459-468.2003
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发表时间：
2003-05-01
期刊：
影响因子：
--
通讯作者：
Dea, S
中科院分区：
文献类型：
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作者：
Bouh, KCS;Shareck, F;Dea, S
猪肺炎支原体P46和P65蛋白是两种携带种属特异性抗原决定簇的膜蛋白。基于参考菌株ATCC 25934的基因组序列，设计引物用于PCR扩增编码完整P46（1，260 bp）和P65（1，803 bp）以及N-末端截短的P65（1，200 bp）的基因。这些引物对M.由于不能用通常定殖于猪呼吸道的其它支原体种获得DNA扩增子，因此，然后将两个扩增的基因克隆到pGEX-4 T-1载体中，以在大肠杆菌细胞中表达为与谷胱甘肽S-转移酶（GST）的重组融合蛋白。在产生表达构建体之前，TGA无义密码子，特别是用于色氨酸残基的M.通过PCR定向诱变将猪肺炎支原体转化为TGG密码子。通过IPTG（异丙基-β-D-硫代半乳糖苷）诱导后，通过超声破碎转化的细胞回收GST-P46和GST-P65（c）重组融合蛋白，通过亲和层析纯化，然后用凝血酶切割以释放P46和P65（c）部分。富集的E.用大肠杆菌表达的P46和P65（c）蛋白免疫雌性BALB/c小鼠，制备抗P46和抗P65（c）单克隆抗体。用同源株制备的细胞裂解液进行蛋白质印迹，证实所获得的单克隆抗体的多肽特异性。抗P46和抗P65单克隆抗体与另外两种M.猪肺炎链球菌参考菌株（ATCC 25095和J菌株）和魁北克田间菌株（已在培养物中分离）表明，针对两种膜蛋白获得的MAb针对高度保守的物种特异性表位。未证明与检测的其他支原体种属具有反应性。提示地方性肺炎的临床体征和病变在无特定病原体的猪中重现，这些猪已经用M的强毒魁北克田间菌株（IAF-DM 9827）进行了肠道接种。下丘脑抗P46和抗P65 c单克隆抗体均能有效地用间接免疫荧光法和间接免疫过氧化物酶法检测M.分别用猪的冷冻和福尔马林固定、石蜡包埋的肺切片（在6- 7周的观察期后处死），
The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 lip) and N-terminally truncated P65, (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-beta-D-thiogalactopyranoside), both GST-P46 and GST-P65(c) recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65(c) moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65(c) monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65, MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65c MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.