虎源猫泛白细胞减少症病毒VP2蛋白的原核表达及应用研究
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摘要
猫泛白细胞减少症是由猫泛白细胞减少症病毒(Feline panleukopenia virus,FPV)引起猫科动物以高热、呕吐、脱水、白细胞严重减少和出血性肠炎为主要特征的急性、高度接触性传染病。在自然条件下,FPV可感染虎、豹、狮、水貂、浣熊等多种食肉类动物,具有很高的致死率,严重威胁圈养及野生虎等大型猫科动物的养殖和保护,迫切需要建立能够同时大批量检测多种动物FPV感染的通用诊断方法。
VP2蛋白为FPV主要结构蛋白,是FPV衣壳的主要成分,暴露在衣壳蛋白表面,为FPV的主要免疫保护性抗原蛋白,能诱导机体产生中和抗体,是研究FPV诊断抗原制备的首选对象。目前,尚未见有利用原核表达系统完整表达VP2基因的报道。
本研究利用CRFK细胞对某东北某虎林园发生传染性出血性肠炎的老虎粪便进行了病毒的分离和鉴定,对分离的虎源FPV保护性抗原VP2蛋白全长基因进行克隆与遗传变异分析。在此基础上,利用原核表达载体PGEX-6P-1在BL21宿主菌中表达该基因。以表达的重组VP2蛋白作为抗原,通过建立检测家猫FPV自然感染抗体的间接ELISA方法来研究2种属来源FPV VP2蛋白的抗原特性变化及该蛋白作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。基于VP2蛋白为检测FPV优势诊断抗原特性,利用纯化的重组VP2蛋白替代传统的全病毒成份作为诊断抗原,以辣根过氧化物酶(HRP)标记的SPA作为广谱第二抗体,初步建立适用于2种猫科动物猫泛白细胞减少症血清抗体检测的SPA-ELISA方法。用建立的SPA-ELISA方法对临床30份猫血清样本、86份虎血清样本进行检测,同时与HI、间接ELISA方法进行比较。在此基础上,以纯化的重组VP2蛋白为免疫原,FPV全病毒作为筛选抗原,运用淋巴细胞杂交瘤技术,制备虎源FPV VP2蛋白特异性单克隆抗体。结果如下：
利用CRFK细胞从老虎粪便中成功分离出强毒株FPV-HLJ2,测得该毒株VP2基因全长为1755个核苷酸,编码584个氨基酸。序列分析表明该病毒与虎源FPV-HLJ株核苷酸同源性为100%,实为同一毒株。
利用原核表达系统实现了虎源FPVVP2基因的表达,SDS-PAGE和Western blot显示表达产物以包涵体形式存在,大小约为84 ku,并具有抗原性。其中目的蛋白58 ku, GST标签蛋白26ku。在家猫猫泛白细胞减少症间接ELISA抗体检测法的应用中,该外源表达蛋白亦显示出很好的抗原特异性,VP2蛋白上一些位点氨基酸的变异并未影响FPV抗原性,表达的重组VP2蛋白可以作为诊断抗原用于家猫FPV抗体的间接ELISA检测,初步证明该蛋白具有作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。
以纯化的重组VP2蛋白替代传统的全病毒成份作为诊断抗原,建立了SPA-ELISA方法,确定了检测2种猫科动物FPV抗体的SPA-ELISA最佳操作程序,批内及批间重复试验均显示变异系数小于10%。对临床30份猫血清样本、86份虎血清样本的对比检测结果显不,SPA-ELISA方法检出率较高,大于HI法的检出率,与间接ELISA法接近。三种检测方法检测猫血清的总体符合率为96.7%。在虎血清检测中,SPA-ELISA方法的阳性检出率亦高于HI方法,二者总体符合率为94.2%。
以重组VP2蛋白作为免疫抗原,用FPV全病毒作为筛选抗原,采用传统的杂交瘤技术,经过间接ELISA方法筛选和有限稀释法亚克隆,最终获得了1株可稳定分泌虎源FPVVP2蛋白特异性单克隆抗体的杂交瘤细胞株3C4。间接ELISA方法测定3C4株单抗腹水效价为1：12800,亚类鉴定为IgG2a类型,间接免疫荧光鉴定试验显示,该株单抗能使接种虎源FPV的CRFK细胞产生亮绿色荧光,而免疫印迹试验显示该株单抗未见特异性反应带。
本研究初步明确了2种属来源的FPV抗原性差异,实现了FPV VP2蛋白的初步实践应用。基于FPV VP2蛋白建立的SPA-ELISA诊断方法,解决了目前虎等猫科动物FPV抗体检测方法非常单一、不能大批量检测等问题,有望用于豹、狮、熊猫、豹猫、浣熊、水貂、猞猁等更多种类动物血清猫泛白细胞减少症病毒抗体的检测。对实现适用于更多种圈养及野生猫科动物等猫瘟热病的快速诊断、疫情监测、流行病学调查和免疫抗体效价测定具有重要意义。基于FPV VP2蛋白制备的FPV VP2蛋白特异性单克隆抗体,为FPV、CPV和MEV诊断及分子生物学研究提供一种更为敏感和特异的试剂。
Feline Panleukopenia is an acute and highly contagious viral disease caused by Feline Panleukopenia virus (FPV). The main characteristics of this disease are high fever, vomiting, clehydration, serious decrease of the white blood cells and haemorrhagic enteritis. Under the natural conditions, multiple kinds of carnivores, such as tigers, leopards, lions minks and raccoons, can be infected by FPV with a high mortality rate; and which has posed a serious threat to the feeding and protection of big cats including both domestic and wild animals of the Felinedae. Therefore, a common diagnostic method is badly needed to set up to test FPV of various animals simultaneously on a large scale.
VP2 protein is the main component of the capsid and major structure protein of FPV which is exposed to the surface the capsid protein. Furthermore, it is also the main immune protein antigen which is able to induce the body to produce antibodies. Thus, VP2 protein is the best for the research of the preparation of FPV diagnostic antigens. At the moment, there haven't researches of whole VP2 gene expression by prokaryotic expression system been reported.
FPV strain was isolated from drops of diarrhea tigers from a wildlife zoo in China and identified by culturing on the CRFK cell line. VP2 gene of FPV was cloned and analyzed. The amplified VP2 gene was subcloned into the prokaryotic expressing vector pGEX-6p-1, and then transferred into E.coli BL21(DE3) for expression under IPTG treatment. The purified recombinant VP2 protein was analyzed by Western Blot. An indirect enzyme-linked immunosorbent assay (ELISA) for target antibodies detecting was developed by the purified recombinant VP2 protein to study its characteristics of Common Diagnostic Antigen of VP2 protein. Since the purified recombinant VP2 protein is a superior diagnostic antigen, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of cats and tigers.30 clinical serumal samples of domestic cats and 86 serumal samples of tigers were tested by SPA-ELISA, HI and indirect ELISA to compare total coincidence ratio. And then, monoclonal antibody (McAb) against recombinant VP2 protein of tiger feline panleukopenia virus were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger FPV. The results are as follows:
The FPV-HLJ2 was isolated from drops of diarrhea tiger by culturing on the CRFK cell line successfully；sequencing analysis of VP2 gene showed that the full length of VP2 gene was 1755 bp and encoded 584 amino acids, and FPV-HLJ2 isolate shares 100% identity with FPV-HLJ isolate (Feline Panleukopenia Virus tiger strain), while the host tropism amino acid changes occurred in the same point.
VP2 gene was highly effectively expressed in E. coli. SDS-PAGE and Western blotting assays revealed that the fusion protein of about 84 ku was obtained from the prokaryotic expression system, including a 58 ku VP2 protein and a 26 ku GST-tag protein, and had good immunoreactivity. The indirect ELISA assay indicated a good antigenic specificity of the expressed VP2 protein and the feasibility to be used as diagnostic antigen for the diagnosis of FPV infection in cats. Some variation of sites AA on the VP2 Protein didn't affect the antigenic nature of FPV. The potential of recombinant VP2 protein to be used as Common Diagnostic Antigen.for the diagnosis of FPV infection in felids was revealed.
Based on the purified recombinant VP2 protein of tiger FPV, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of domestic cats and tigers. The assay was optimized. Variation coefficient of intraassay and interassay about SPA-ELISA were all less than 10%. The comparing detection among the 30 serumal samples of domestic cats and 86 serumal samples of tigers showed that the detection rate by SPA-ELISA was higher than HI, and the result was close to indirect ELISA. The total coincidence ratio of the three detecting methods were 96.7% by detecting 30 serum samples of cats. The coincidence ratio of the SPA-ELISA and HI was 94.2% by detecting 86 serumal samples of tigers. All these results indicated that SPA-ELISA could be a good method for serological detection of FPV antibodies in many species of felines.
Monoclonal antibody 3C4 against recombinant VP2 protein of tiger FPV were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger feline panleukopenia virus. The antibody titers of ascites for the hybridoma lines were 1:12800. The subtype of monoclonal antibodies was determined as IgG2a. Indirect immunofluorescent test indicated that the monoclonal antibody could specifically react with FPV, and it may be used as a valuable tools for diagnosis of FPV.
The difference between FPVs from two different kinds of mammals was initially known through the study, and the preliminary use of FPV VP2 Protein come true. The way of SPA-ELISA based on FPV VP2 protein, solving the problem that the present detections are too simple or can't detect a large amount of samples at one time, is likely to be used to test FPV antibodies in animals' serum such as leopards, lions, pandas, ocelots, raccoons, minks, lynx, etc. There will be great significance in realizing quick diagnosis, epidemic observation, epizootic research and detection of antibody potency. The neutralizing monoclonal antibody of FPV VP2 Protein based on FPV VP2 provides a more sensitive and different reagent for FPV, CPV and MEV diagnosis.
VP2蛋白为FPV主要结构蛋白,是FPV衣壳的主要成分,暴露在衣壳蛋白表面,为FPV的主要免疫保护性抗原蛋白,能诱导机体产生中和抗体,是研究FPV诊断抗原制备的首选对象。目前,尚未见有利用原核表达系统完整表达VP2基因的报道。
本研究利用CRFK细胞对某东北某虎林园发生传染性出血性肠炎的老虎粪便进行了病毒的分离和鉴定,对分离的虎源FPV保护性抗原VP2蛋白全长基因进行克隆与遗传变异分析。在此基础上,利用原核表达载体PGEX-6P-1在BL21宿主菌中表达该基因。以表达的重组VP2蛋白作为抗原,通过建立检测家猫FPV自然感染抗体的间接ELISA方法来研究2种属来源FPV VP2蛋白的抗原特性变化及该蛋白作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。基于VP2蛋白为检测FPV优势诊断抗原特性,利用纯化的重组VP2蛋白替代传统的全病毒成份作为诊断抗原,以辣根过氧化物酶(HRP)标记的SPA作为广谱第二抗体,初步建立适用于2种猫科动物猫泛白细胞减少症血清抗体检测的SPA-ELISA方法。用建立的SPA-ELISA方法对临床30份猫血清样本、86份虎血清样本进行检测,同时与HI、间接ELISA方法进行比较。在此基础上,以纯化的重组VP2蛋白为免疫原,FPV全病毒作为筛选抗原,运用淋巴细胞杂交瘤技术,制备虎源FPV VP2蛋白特异性单克隆抗体。结果如下：
利用CRFK细胞从老虎粪便中成功分离出强毒株FPV-HLJ2,测得该毒株VP2基因全长为1755个核苷酸,编码584个氨基酸。序列分析表明该病毒与虎源FPV-HLJ株核苷酸同源性为100%,实为同一毒株。
利用原核表达系统实现了虎源FPVVP2基因的表达,SDS-PAGE和Western blot显示表达产物以包涵体形式存在,大小约为84 ku,并具有抗原性。其中目的蛋白58 ku, GST标签蛋白26ku。在家猫猫泛白细胞减少症间接ELISA抗体检测法的应用中,该外源表达蛋白亦显示出很好的抗原特异性,VP2蛋白上一些位点氨基酸的变异并未影响FPV抗原性,表达的重组VP2蛋白可以作为诊断抗原用于家猫FPV抗体的间接ELISA检测,初步证明该蛋白具有作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。
以纯化的重组VP2蛋白替代传统的全病毒成份作为诊断抗原,建立了SPA-ELISA方法,确定了检测2种猫科动物FPV抗体的SPA-ELISA最佳操作程序,批内及批间重复试验均显示变异系数小于10%。对临床30份猫血清样本、86份虎血清样本的对比检测结果显不,SPA-ELISA方法检出率较高,大于HI法的检出率,与间接ELISA法接近。三种检测方法检测猫血清的总体符合率为96.7%。在虎血清检测中,SPA-ELISA方法的阳性检出率亦高于HI方法,二者总体符合率为94.2%。
以重组VP2蛋白作为免疫抗原,用FPV全病毒作为筛选抗原,采用传统的杂交瘤技术,经过间接ELISA方法筛选和有限稀释法亚克隆,最终获得了1株可稳定分泌虎源FPVVP2蛋白特异性单克隆抗体的杂交瘤细胞株3C4。间接ELISA方法测定3C4株单抗腹水效价为1：12800,亚类鉴定为IgG2a类型,间接免疫荧光鉴定试验显示,该株单抗能使接种虎源FPV的CRFK细胞产生亮绿色荧光,而免疫印迹试验显示该株单抗未见特异性反应带。
本研究初步明确了2种属来源的FPV抗原性差异,实现了FPV VP2蛋白的初步实践应用。基于FPV VP2蛋白建立的SPA-ELISA诊断方法,解决了目前虎等猫科动物FPV抗体检测方法非常单一、不能大批量检测等问题,有望用于豹、狮、熊猫、豹猫、浣熊、水貂、猞猁等更多种类动物血清猫泛白细胞减少症病毒抗体的检测。对实现适用于更多种圈养及野生猫科动物等猫瘟热病的快速诊断、疫情监测、流行病学调查和免疫抗体效价测定具有重要意义。基于FPV VP2蛋白制备的FPV VP2蛋白特异性单克隆抗体,为FPV、CPV和MEV诊断及分子生物学研究提供一种更为敏感和特异的试剂。
Feline Panleukopenia is an acute and highly contagious viral disease caused by Feline Panleukopenia virus (FPV). The main characteristics of this disease are high fever, vomiting, clehydration, serious decrease of the white blood cells and haemorrhagic enteritis. Under the natural conditions, multiple kinds of carnivores, such as tigers, leopards, lions minks and raccoons, can be infected by FPV with a high mortality rate; and which has posed a serious threat to the feeding and protection of big cats including both domestic and wild animals of the Felinedae. Therefore, a common diagnostic method is badly needed to set up to test FPV of various animals simultaneously on a large scale.
VP2 protein is the main component of the capsid and major structure protein of FPV which is exposed to the surface the capsid protein. Furthermore, it is also the main immune protein antigen which is able to induce the body to produce antibodies. Thus, VP2 protein is the best for the research of the preparation of FPV diagnostic antigens. At the moment, there haven't researches of whole VP2 gene expression by prokaryotic expression system been reported.
FPV strain was isolated from drops of diarrhea tigers from a wildlife zoo in China and identified by culturing on the CRFK cell line. VP2 gene of FPV was cloned and analyzed. The amplified VP2 gene was subcloned into the prokaryotic expressing vector pGEX-6p-1, and then transferred into E.coli BL21(DE3) for expression under IPTG treatment. The purified recombinant VP2 protein was analyzed by Western Blot. An indirect enzyme-linked immunosorbent assay (ELISA) for target antibodies detecting was developed by the purified recombinant VP2 protein to study its characteristics of Common Diagnostic Antigen of VP2 protein. Since the purified recombinant VP2 protein is a superior diagnostic antigen, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of cats and tigers.30 clinical serumal samples of domestic cats and 86 serumal samples of tigers were tested by SPA-ELISA, HI and indirect ELISA to compare total coincidence ratio. And then, monoclonal antibody (McAb) against recombinant VP2 protein of tiger feline panleukopenia virus were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger FPV. The results are as follows:
The FPV-HLJ2 was isolated from drops of diarrhea tiger by culturing on the CRFK cell line successfully；sequencing analysis of VP2 gene showed that the full length of VP2 gene was 1755 bp and encoded 584 amino acids, and FPV-HLJ2 isolate shares 100% identity with FPV-HLJ isolate (Feline Panleukopenia Virus tiger strain), while the host tropism amino acid changes occurred in the same point.
VP2 gene was highly effectively expressed in E. coli. SDS-PAGE and Western blotting assays revealed that the fusion protein of about 84 ku was obtained from the prokaryotic expression system, including a 58 ku VP2 protein and a 26 ku GST-tag protein, and had good immunoreactivity. The indirect ELISA assay indicated a good antigenic specificity of the expressed VP2 protein and the feasibility to be used as diagnostic antigen for the diagnosis of FPV infection in cats. Some variation of sites AA on the VP2 Protein didn't affect the antigenic nature of FPV. The potential of recombinant VP2 protein to be used as Common Diagnostic Antigen.for the diagnosis of FPV infection in felids was revealed.
Based on the purified recombinant VP2 protein of tiger FPV, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of domestic cats and tigers. The assay was optimized. Variation coefficient of intraassay and interassay about SPA-ELISA were all less than 10%. The comparing detection among the 30 serumal samples of domestic cats and 86 serumal samples of tigers showed that the detection rate by SPA-ELISA was higher than HI, and the result was close to indirect ELISA. The total coincidence ratio of the three detecting methods were 96.7% by detecting 30 serum samples of cats. The coincidence ratio of the SPA-ELISA and HI was 94.2% by detecting 86 serumal samples of tigers. All these results indicated that SPA-ELISA could be a good method for serological detection of FPV antibodies in many species of felines.
Monoclonal antibody 3C4 against recombinant VP2 protein of tiger FPV were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger feline panleukopenia virus. The antibody titers of ascites for the hybridoma lines were 1:12800. The subtype of monoclonal antibodies was determined as IgG2a. Indirect immunofluorescent test indicated that the monoclonal antibody could specifically react with FPV, and it may be used as a valuable tools for diagnosis of FPV.
The difference between FPVs from two different kinds of mammals was initially known through the study, and the preliminary use of FPV VP2 Protein come true. The way of SPA-ELISA based on FPV VP2 protein, solving the problem that the present detections are too simple or can't detect a large amount of samples at one time, is likely to be used to test FPV antibodies in animals' serum such as leopards, lions, pandas, ocelots, raccoons, minks, lynx, etc. There will be great significance in realizing quick diagnosis, epidemic observation, epizootic research and detection of antibody potency. The neutralizing monoclonal antibody of FPV VP2 Protein based on FPV VP2 provides a more sensitive and different reagent for FPV, CPV and MEV diagnosis.
引文
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