鸡毒霉形体HS株TM-1和mgc3基因的表位预测及原核表达
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摘要
鸡毒霉形体(Mycoplasma gallisepticum，MG)是鸡慢性呼吸道疾病的病原菌，世界各地均有MG的感染和流行，虽然不直接引起禽的大规模死亡，但疾病的慢性、迁延性流行过程可引起产蛋率下降、孵化率降低、出栏期延长及饲料利用率降低，每年给世界养禽业造成了巨大的经济损失。
目前MG的诊断方法主要有病原的分离鉴定、血清平板凝集试验、PCR和EHSA等方法，但上述方法由于耗时或操作复杂或需要昂贵仪器等缺点，难以在基层推广应用，因此MG新型的快速诊断也成为该病急需解决的问题之一。胶体金免疫层析快速检测MG的方法简便、快速、直观，但是研制抗不同抗原表位的单克隆抗体是该方法建立的重要前提。本研究对MG菌体保护性蛋白TM-1和mgc3基因的表达及抗原表位进行了探讨，旨在为ELISA方法、胶体金免疫层析法快速检测方法及基因工程抗原疫苗的研制提供理论依据，主要展开了以下研究。
1．鸡毒霉形体保护性抗原基因TM-1和mgc3的克隆
参照NCBI收录的TM-1和mgc3基因序列设计引物，以MG基因组为模板，扩增出TM-1和mgc3，经测序，所获得的TM-1基因为819bp；mgc3基因为3186bp。
2．TM-1和mgc3基因序列的分析及抗原表位的预测
TM-1基因测序结果在NCBI数据库中BLAST比对，与S6株同源性为99％，其中103-134bp和135-166bp为重复序列，抗原表位及二级结构的预测参数综合分析，该重复区具有强抗原表位活性；与R株有两个同源性序列，同源性分别为91％和93％。对mgc3基因的全基因序列BLAST比对，得到三条同源性达99％的序列，此外还有三条序列只与mgc3基因的1-1300bp具有同源性，分别是99％、98％和91％。选取了HS株的mgc3基因1000-3186bp的区域进行抗原表位软件预测，得到1142-1440bp、1704-2169bp、2165-2521bp和2507-2835bp区域具有较强的抗原活性。它们不仅亲水性、表面可及性、抗原性参数较高，而且每段序列的上下游引物都包含了该区的所有突变位点；MG-HS株的mgc3基因中还有10个编码色氨酸TGA的位点。
3．鸡毒霉形体TM-1和mgc3基因抗原表位区的原核表达及其活性鉴定
将经过分析的含有高免疫活性的目的片段亚克隆到表达载体pGEX-KG，经转化、诱导后获得表达蛋白，SDS-PAGE分析表明融合表达的蛋白分子量分别约为55KDa、37KDa和44 KDa。Western blot印迹证明表达蛋白均具有免疫学活性。说明表达蛋白可用于免疫诊断和也为基因工程亚单位疫苗的研制奠定了基础。
Mycoplasma gallisepticum is the aetiologic agent of chronic respiratory disease inchickens.The diseases caused by MG in chickens break out around the world, whichmainly led to high unhealthy chicken rate and egg production of layer dropped, feedconversion efficiency reduced. Infection of MG don't cause large scale death of chickendirectly but it lead to heavier economy loss in chicken firm indirectly all of the world.
At present, the main of normal methods for MG-diagnosis, such as the isolation andidentification of thallus culture, serum plate agglutination (SPA), polymerase chainreaction (PCR) and enzyme linked immunosorbent assay(ELISA), are limited in spotapplication because of their time-consuming, complicated manipulation or requitingexpensive instruments. The development of rapid diagnosis for MG has become one ofthe problems need to be solved urgently. Gold-immunochromatographic assay (GICA) isconvenient, rapid and direct-viewing. However, the preparation of monoclonal antibody(McAb) against the different epitopes is the prerequisite of GICA. The result of this studywill provide strong theoretical support for the development of the detceted method GICAand ELISA and the preparation of genetically engineering vaccine. The main contents andresults of research are summarized as following:
1. Cloning of TM-1 and mgc3 genes coding the protective antigen of Mycoplasmagallisepticum
According to published TM-1 and the mgc3 gene sequences MG in Genbank, primerswith restriction sites were designed and synthesized. The expectant fragments wereamplified from strain HS by common PCR.The sequence analysis showed that TM-landthe mgc3 genes consisted of 819 base pair(bp) and 3186 base pair respectively.
2. Sequence analysis and epitope prediction of TM-1 and mgc3 by biologicalsoftware.
The comparison of the TM-1 gene sequence amplified from MG-HS strain to othermycoplasma revealed that their homology were up to 99% with MG-S6 strain. The repeat sequences of TM-1 gene exists as the 103～134bp and 135～166bp in MG-HS strain. Itwas predicted that this epitope have strong antigenically by secondary structure analysis.A striking homology of DNA sequences between MG-HS and MG-R is up to 91% and93 % the sequence of TM-1 gene. It is confirmed that there are three gene sequenceswith high homology up to 99% by blasting of the entire mgc3 gene sequence betweenMG-HS and MG-R. Moreover there are three sequences present homology in front 1100bpof mgc3 gene between MG-HS and MG-S6, their homology is 99%, 98% and 91%respectively. So the 1000-3186bp sequences of mgc3 gene have been analysised andpredictied by software, we found four epitope regions, they are 142～1440bp、1704～2169bp、2165～2521bp and 2507～2835bp respectively. It is prognosed that they arestronge hydrophilic, surface accessible, antigenic. There are 10 TGA codons encodingtryptophan in mgc3 gene sequence.
3. The expression of MG-HS TM-1 and epitopes of mgc3 in E.coil BL21 (DE3) andthe identification of biologic activity of the expressed protein
The fragments of TM-1 and mgc3-P1, ngc3-P1 were subcloned into prokaryoticexpressing vector pGEX-KG and expressed induced by IPTG in E. coli B121. Analyzed bySDS-PAGE and western blotting, 29Ku, MGC3-P1 and MGC3-P2 fusion proteinspresented immunogenicity with 55 KDa,37 KDa and 44KDa proteins respectively.
目前MG的诊断方法主要有病原的分离鉴定、血清平板凝集试验、PCR和EHSA等方法，但上述方法由于耗时或操作复杂或需要昂贵仪器等缺点，难以在基层推广应用，因此MG新型的快速诊断也成为该病急需解决的问题之一。胶体金免疫层析快速检测MG的方法简便、快速、直观，但是研制抗不同抗原表位的单克隆抗体是该方法建立的重要前提。本研究对MG菌体保护性蛋白TM-1和mgc3基因的表达及抗原表位进行了探讨，旨在为ELISA方法、胶体金免疫层析法快速检测方法及基因工程抗原疫苗的研制提供理论依据，主要展开了以下研究。
1．鸡毒霉形体保护性抗原基因TM-1和mgc3的克隆
参照NCBI收录的TM-1和mgc3基因序列设计引物，以MG基因组为模板，扩增出TM-1和mgc3，经测序，所获得的TM-1基因为819bp；mgc3基因为3186bp。
2．TM-1和mgc3基因序列的分析及抗原表位的预测
TM-1基因测序结果在NCBI数据库中BLAST比对，与S6株同源性为99％，其中103-134bp和135-166bp为重复序列，抗原表位及二级结构的预测参数综合分析，该重复区具有强抗原表位活性；与R株有两个同源性序列，同源性分别为91％和93％。对mgc3基因的全基因序列BLAST比对，得到三条同源性达99％的序列，此外还有三条序列只与mgc3基因的1-1300bp具有同源性，分别是99％、98％和91％。选取了HS株的mgc3基因1000-3186bp的区域进行抗原表位软件预测，得到1142-1440bp、1704-2169bp、2165-2521bp和2507-2835bp区域具有较强的抗原活性。它们不仅亲水性、表面可及性、抗原性参数较高，而且每段序列的上下游引物都包含了该区的所有突变位点；MG-HS株的mgc3基因中还有10个编码色氨酸TGA的位点。
3．鸡毒霉形体TM-1和mgc3基因抗原表位区的原核表达及其活性鉴定
将经过分析的含有高免疫活性的目的片段亚克隆到表达载体pGEX-KG，经转化、诱导后获得表达蛋白，SDS-PAGE分析表明融合表达的蛋白分子量分别约为55KDa、37KDa和44 KDa。Western blot印迹证明表达蛋白均具有免疫学活性。说明表达蛋白可用于免疫诊断和也为基因工程亚单位疫苗的研制奠定了基础。
Mycoplasma gallisepticum is the aetiologic agent of chronic respiratory disease inchickens.The diseases caused by MG in chickens break out around the world, whichmainly led to high unhealthy chicken rate and egg production of layer dropped, feedconversion efficiency reduced. Infection of MG don't cause large scale death of chickendirectly but it lead to heavier economy loss in chicken firm indirectly all of the world.
At present, the main of normal methods for MG-diagnosis, such as the isolation andidentification of thallus culture, serum plate agglutination (SPA), polymerase chainreaction (PCR) and enzyme linked immunosorbent assay(ELISA), are limited in spotapplication because of their time-consuming, complicated manipulation or requitingexpensive instruments. The development of rapid diagnosis for MG has become one ofthe problems need to be solved urgently. Gold-immunochromatographic assay (GICA) isconvenient, rapid and direct-viewing. However, the preparation of monoclonal antibody(McAb) against the different epitopes is the prerequisite of GICA. The result of this studywill provide strong theoretical support for the development of the detceted method GICAand ELISA and the preparation of genetically engineering vaccine. The main contents andresults of research are summarized as following:
1. Cloning of TM-1 and mgc3 genes coding the protective antigen of Mycoplasmagallisepticum
According to published TM-1 and the mgc3 gene sequences MG in Genbank, primerswith restriction sites were designed and synthesized. The expectant fragments wereamplified from strain HS by common PCR.The sequence analysis showed that TM-landthe mgc3 genes consisted of 819 base pair(bp) and 3186 base pair respectively.
2. Sequence analysis and epitope prediction of TM-1 and mgc3 by biologicalsoftware.
The comparison of the TM-1 gene sequence amplified from MG-HS strain to othermycoplasma revealed that their homology were up to 99% with MG-S6 strain. The repeat sequences of TM-1 gene exists as the 103～134bp and 135～166bp in MG-HS strain. Itwas predicted that this epitope have strong antigenically by secondary structure analysis.A striking homology of DNA sequences between MG-HS and MG-R is up to 91% and93 % the sequence of TM-1 gene. It is confirmed that there are three gene sequenceswith high homology up to 99% by blasting of the entire mgc3 gene sequence betweenMG-HS and MG-R. Moreover there are three sequences present homology in front 1100bpof mgc3 gene between MG-HS and MG-S6, their homology is 99%, 98% and 91%respectively. So the 1000-3186bp sequences of mgc3 gene have been analysised andpredictied by software, we found four epitope regions, they are 142～1440bp、1704～2169bp、2165～2521bp and 2507～2835bp respectively. It is prognosed that they arestronge hydrophilic, surface accessible, antigenic. There are 10 TGA codons encodingtryptophan in mgc3 gene sequence.
3. The expression of MG-HS TM-1 and epitopes of mgc3 in E.coil BL21 (DE3) andthe identification of biologic activity of the expressed protein
The fragments of TM-1 and mgc3-P1, ngc3-P1 were subcloned into prokaryoticexpressing vector pGEX-KG and expressed induced by IPTG in E. coli B121. Analyzed bySDS-PAGE and western blotting, 29Ku, MGC3-P1 and MGC3-P2 fusion proteinspresented immunogenicity with 55 KDa,37 KDa and 44KDa proteins respectively.
引文
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