猪脑心肌炎病毒非结构蛋白3AB和3ABC基因的表达与3AB单克隆抗体的制备
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摘要
脑心肌炎病毒(Encephalomyocarditis virus,EMCV)是一种重要的人畜共患病病原。EMCV感染最广泛和最严重的动物是猪,感染后仔猪可造成急性心肌炎等实质器官的广泛病理损伤甚至发生急性死亡;感染母猪可导致怀孕母猪流产、产死胎、弱胎、木乃伊胎等繁殖障碍。目前国内外该病的病原学诊断和血清学检测方法都是针对全病毒或结构蛋白,尚无针对非结构蛋白方面的检测方法的研究。EMCV预防临床最常用还是灭活苗,建立检测非结构蛋白的抗体的方法,可用于区别疫苗免疫和自然感染。EMCV和口蹄疫病毒(FMDV)同属微RNA病毒科,其基因结构类似。许多研究证明FMDV 3AB和3ABC ELISA方法能有效检测FMDv自然感染水平,但在EMCV研究方面尚无类似的报道。本研究通过原核和真核表达系统表达EMCV非结构蛋白3AB和3ABc,并制备3AB的单克隆抗体,主要内容包括:
1.EMCV 3AB和3ABc基因原核表达与抗体制备:首先通过PCR方法从重组质粒pUC18-EMCV中分别扩增得到含完整阅读框的3AB和3ABC基因,克隆至原核高效表达载体pGEX-6p-1中,在E.coli BL_(21)(DE_3)细菌中分别成功表达了重组蛋白GST-3AB和GST-3ABC,大小分别为39kDa和62kDa。由于EMCV 3C蛋白为蛋白质水解酶,易从3ABC上解离下来,故部分GST-3ABC蛋白降解为GST-3AB和3C(22kDa)。取包涵体纯化的GST-3AB蛋白免疫清洁级小鼠,制备得到3AB蛋白的多克隆抗体。Western bolt结果显示,该抗体可与重组3AB和3ABC蛋白发生特异性反应。
2.杆状病毒表达EMCV 3AB和3ABC基因:先将目的基因分别克隆至杆状病毒转座载体pFastBac~(TM)中,提取阳性克隆质粒转化至DH_(10) Bac感受态细菌,通过蓝白斑筛选和PCR鉴定,将阳性重组杆状病毒穿梭载体经脂质体介导转染Sf9细胞获得重组杆状病毒,并经IFA和Western blot鉴定。试验结果显示两个目的蛋白在昆虫细胞中获得成功表达,重组3AB分子量约16kDa,重组3ABC蛋白仍然解离成3AB和3C。
3.EMCV 3AB单克隆抗体制备:用纯化的GST-3AB蛋白免疫BALB/c小鼠并通过杂交瘤细胞技术制备其单克隆抗体,经间接ELISA筛选阳性的杂交瘤细胞,获得1株能稳定分泌抗3AB蛋白抗体的杂交瘤细胞株,将其命名为2D12,其亚类测定为IgG1/κ。Weatern Blot和间接免疫荧光试验证明该单抗能特异性识别3AB蛋白。成功获得了针对EMCV 3AB的特异性单抗。
本研究通过基因工程手段成功表达获得EMCV非结构蛋白3AB和3ABC,并制备获得抗3AB的单多克隆抗体,为开展相关的非结构蛋白功能及血清学诊断方法研究奠定了重要基础。
Encephalomyocarditis virus (EMCV) is a new kind of zoonoses virus. which causes acute myocarditis and sudden death in preweaning piglets or severe reproductive failure in sows. Most of the methods for serological survey of disease have been developed to detect the antibody to the whole virus or structural proteins but not non-structural protein. FMDV and EMCV were belong to picornaviridae, they have similar genome structure. It has reported that 3AB and 3ABC ELISA could be used to differentiate vaccinated animals from FMDV infected animals. In this study 3AB and 3ABC genes of EMCV were expressed in prokaryotic expression system and baculovirus expression system, respectively, and one monoclonal antibody against 3AB was prepared. The details as follow:
1. Expression of EMCV 3AB and 3ABC genes in prokaryotic expression system and prepare polyclonal antibodies against 3AB protein
NSP 3AB and 3ABC genes of Encephalomyocarditis virus (EMCV) was amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and recombinant proteins 3AB and 3ABC were expressed in E. coli with 36kDa and 62kDa sucessfully. And most of recombinant proteins GST-3ABC were cleaved into GST-3AB and 3C, it may due to the effection of 3C as proteinase. Then mices were immunized by purified recombinant 3AB protein of inclusion-body, the serum was collected two weeks after 3 times immunization. The result of Western blot show that polyclonal antibodies against 3AB protein could specificially react with GST-3AB and GST-3ABC proteins.
2. Expression and identification of 3AB and 3ABC genes of EMCV in baculovirus expression system
In this experiment, the 3AB and 3ABC genes of EMCV were cloned into baculovirus expression vector pFastBac~(TM), and confirmed by enzyme digestion. Then, recombinant plasmid was transformed to DH10 Bac competent cells, and the recombinant Bacmid-3AB and Bacmid-3ABC plasmids were screened by blue-white colony and PCR. After transfection of the purified plasmids into Sf9 cells respectively, the recombinant baculoviruses were obtained. Expression of the 3AB gene of EMCV was confirmed by indirect immunofluorescent assay (IFA), SDS-PAGE and Western-blot. The expressed 3AB gene product had a molecular weight of 16kDa and could be recognized by the antibody to 3AB protein of EMCV. The results showed that the 3AB protein was efficiently expressed in the recombinant baculovirus. As same as results of prokaryotic expression the recombinant 3ABC protein was also cleaved into 3AB and 3C.
3. Preparation and identification of moloclonal antibody against of 3AB protein The Balb/c mices were immunized by purified recombinant 3AB protein which expressed in prokaryotic expression system, and the splenocytes of the immunized mice were fused with murine myeloma cells to produce hybridoma cell line. After subcloning by 3 times, one strain of hybridoma cell line steadily secreting antibodies of 3AB protein was obtained, named 2D12. The McAb belongs to IgG1 /κ. The McAb was confirmed by indirect immunofluorescent assay (IFA) and Western-blot.
EMCV recombinant 3AB and 3ABC proteins, and the McAb against 3AB protein were obtained throught genomic technologies. All the research provides a potential value for structural and functional studies of EMCV non-structural protein and early diagnosis of Encephalomyocarditis virus infection.
1.EMCV 3AB和3ABc基因原核表达与抗体制备:首先通过PCR方法从重组质粒pUC18-EMCV中分别扩增得到含完整阅读框的3AB和3ABC基因,克隆至原核高效表达载体pGEX-6p-1中,在E.coli BL_(21)(DE_3)细菌中分别成功表达了重组蛋白GST-3AB和GST-3ABC,大小分别为39kDa和62kDa。由于EMCV 3C蛋白为蛋白质水解酶,易从3ABC上解离下来,故部分GST-3ABC蛋白降解为GST-3AB和3C(22kDa)。取包涵体纯化的GST-3AB蛋白免疫清洁级小鼠,制备得到3AB蛋白的多克隆抗体。Western bolt结果显示,该抗体可与重组3AB和3ABC蛋白发生特异性反应。
2.杆状病毒表达EMCV 3AB和3ABC基因:先将目的基因分别克隆至杆状病毒转座载体pFastBac~(TM)中,提取阳性克隆质粒转化至DH_(10) Bac感受态细菌,通过蓝白斑筛选和PCR鉴定,将阳性重组杆状病毒穿梭载体经脂质体介导转染Sf9细胞获得重组杆状病毒,并经IFA和Western blot鉴定。试验结果显示两个目的蛋白在昆虫细胞中获得成功表达,重组3AB分子量约16kDa,重组3ABC蛋白仍然解离成3AB和3C。
3.EMCV 3AB单克隆抗体制备:用纯化的GST-3AB蛋白免疫BALB/c小鼠并通过杂交瘤细胞技术制备其单克隆抗体,经间接ELISA筛选阳性的杂交瘤细胞,获得1株能稳定分泌抗3AB蛋白抗体的杂交瘤细胞株,将其命名为2D12,其亚类测定为IgG1/κ。Weatern Blot和间接免疫荧光试验证明该单抗能特异性识别3AB蛋白。成功获得了针对EMCV 3AB的特异性单抗。
本研究通过基因工程手段成功表达获得EMCV非结构蛋白3AB和3ABC,并制备获得抗3AB的单多克隆抗体,为开展相关的非结构蛋白功能及血清学诊断方法研究奠定了重要基础。
Encephalomyocarditis virus (EMCV) is a new kind of zoonoses virus. which causes acute myocarditis and sudden death in preweaning piglets or severe reproductive failure in sows. Most of the methods for serological survey of disease have been developed to detect the antibody to the whole virus or structural proteins but not non-structural protein. FMDV and EMCV were belong to picornaviridae, they have similar genome structure. It has reported that 3AB and 3ABC ELISA could be used to differentiate vaccinated animals from FMDV infected animals. In this study 3AB and 3ABC genes of EMCV were expressed in prokaryotic expression system and baculovirus expression system, respectively, and one monoclonal antibody against 3AB was prepared. The details as follow:
1. Expression of EMCV 3AB and 3ABC genes in prokaryotic expression system and prepare polyclonal antibodies against 3AB protein
NSP 3AB and 3ABC genes of Encephalomyocarditis virus (EMCV) was amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and recombinant proteins 3AB and 3ABC were expressed in E. coli with 36kDa and 62kDa sucessfully. And most of recombinant proteins GST-3ABC were cleaved into GST-3AB and 3C, it may due to the effection of 3C as proteinase. Then mices were immunized by purified recombinant 3AB protein of inclusion-body, the serum was collected two weeks after 3 times immunization. The result of Western blot show that polyclonal antibodies against 3AB protein could specificially react with GST-3AB and GST-3ABC proteins.
2. Expression and identification of 3AB and 3ABC genes of EMCV in baculovirus expression system
In this experiment, the 3AB and 3ABC genes of EMCV were cloned into baculovirus expression vector pFastBac~(TM), and confirmed by enzyme digestion. Then, recombinant plasmid was transformed to DH10 Bac competent cells, and the recombinant Bacmid-3AB and Bacmid-3ABC plasmids were screened by blue-white colony and PCR. After transfection of the purified plasmids into Sf9 cells respectively, the recombinant baculoviruses were obtained. Expression of the 3AB gene of EMCV was confirmed by indirect immunofluorescent assay (IFA), SDS-PAGE and Western-blot. The expressed 3AB gene product had a molecular weight of 16kDa and could be recognized by the antibody to 3AB protein of EMCV. The results showed that the 3AB protein was efficiently expressed in the recombinant baculovirus. As same as results of prokaryotic expression the recombinant 3ABC protein was also cleaved into 3AB and 3C.
3. Preparation and identification of moloclonal antibody against of 3AB protein The Balb/c mices were immunized by purified recombinant 3AB protein which expressed in prokaryotic expression system, and the splenocytes of the immunized mice were fused with murine myeloma cells to produce hybridoma cell line. After subcloning by 3 times, one strain of hybridoma cell line steadily secreting antibodies of 3AB protein was obtained, named 2D12. The McAb belongs to IgG1 /κ. The McAb was confirmed by indirect immunofluorescent assay (IFA) and Western-blot.
EMCV recombinant 3AB and 3ABC proteins, and the McAb against 3AB protein were obtained throught genomic technologies. All the research provides a potential value for structural and functional studies of EMCV non-structural protein and early diagnosis of Encephalomyocarditis virus infection.
引文
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