东毕吸虫病ELISA诊断方法的建立
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摘要
东毕吸虫病(orientobilharziasis)是由多种东毕吸虫(Orientobilharzia spp.)寄生于牛、羊等动物门静脉和肠系膜静脉内而引起的一种血吸虫病。该病主要分布于亚洲和欧洲的一些国家和地区,常呈地方性流行,对畜牧业危害十分严重,同时东毕吸虫的尾蚴可以引起人的尾蚴性皮炎,严重影响人类的健康,是一种非常重要的人兽共患寄生虫病。东毕吸虫病的早期准确诊断是有效的控制该病的发生和蔓延的重要手段,传统的诊断方法主要为病原学检查和免疫学检测,如粪便水洗沉淀法、毛蚴孵化法、间接血凝试验和酶联免疫吸附试验等。然而病原检查检出率低,且虫体感染已过急性期,延误了有效治疗时间而影响了对该病的控制,免疫学检测常用抗原通常为成虫抗原和虫卵抗原,这两种抗原成分复杂、制备成本高、周期长,限制该法的广泛应用。因此,本试验拟利用分子生物学手段,对在血吸虫诊断中起重要作用的信号蛋白14-3-3基因采用RACE技术进行克隆,并制备成重组蛋白,建立间接ELISA诊断方法。
本试验根据GenBankTM上发表的日本血吸虫、曼氏血吸虫和牛血吸虫信号蛋白14-3-3序列,依其保守区设计合成一对引物,以提取的东毕吸虫总RNA为模板,PT-PCR方法扩增信号蛋白14-3-3基因的中间片段,连接pMD18-T载体并转化到TG1宿主菌中,经鉴定,获得阳性克隆,并进行序列分析。根据测得的序列信息,再设计一对引物,利用5’RACE和3’RACE技术分别扩增目的基因的5’端和3’端,鉴定正确后并测序。利用分子生物学软件拼接三段序列得到全长序列信息。
其次,根据东毕吸虫信号蛋白14-3-3全长cDNA序列设计两条加有EcoRⅠ、XhoⅠ酶切位点的引物,利用RT-PCR从东毕吸虫总RNA中扩增信号蛋白14-3-3全长序列,连接pMD18-T载体并转化到TG1宿主菌中,经鉴定及序列分析,获得阳性克隆。阳性质粒pMD18-T-信号蛋白14-3-3和表达载体pET-30a(+),经EcoRⅠ、XhoⅠ双酶切后回收、连接、转化到TG1宿主菌,提取质粒,酶切鉴定正确后,阳性质粒转化到最佳宿主菌BL21(DE3)中,构建其原核重组表达载体pET-30a-信号蛋白14-3-3,经酶切鉴定和DNA序列分析证实,构建的重组质粒pET-30a-信号蛋白14-3-3中含有信号蛋白14-3-3基因,且基因序列和阅读框架均正确。将重组菌株BL21(pET-30a-信号蛋白14-3-3)按1%的量接种到含有Kan(50μg/mL)的LB液体培养基中,37℃振荡培养2.5h,然后用1mmol/L IPTG诱导4h,融合目的蛋白获得高效表达。经Western blot检测,重组菌株表达的融合蛋白能够被东毕吸虫特异性抗体所识别。
以纯化的重组信号蛋白14-3-3作为抗原包被酶标板,通过对间接ELISA各反应条件的优化,确定了最佳反应条件为:最佳封闭液为5%脱脂奶粉,封闭条件为37℃1h;抗原最佳包被浓度为5.0μg/mL;抗原的最佳包被液为50mM pH9.6的碳酸盐缓冲液;最佳血清稀释液为5%脱脂奶粉,血清稀释倍数为100倍;HRP-兔抗羊IgG的最适工作浓度为1:5 000。采用已确立的间接ELISA反应条件,对105份阴性血清的检测结果进行统计学分析,确定了间接ELISA方法判定标准,即OD450值大于等于0.31为阳性,小于0.31为阴性。应用建立的间接ELISA方法对105份阴性血清和65份阳性血清(经剖检法和形态鉴定证实)的检测结果表明,ELISA方法的特异性为91.5%,敏感性为88.6%。而且与肝片吸虫、鹿前后盘吸虫、扩展莫尼茨绦虫和捻转血矛线虫阳性血清均无交叉反应。采用本研究建立的方法,对来自于不同地区的240份绵羊血清进行了检测,结果阳性率为35.3%,与省内的感染情况基本相似。
Adult worms of Orientobilharzia turkestanicum live in the portal veins or intestinal veins of cattle, sheep and other animals and cause orientobilharziasis. It is widely distributed in Asia and several areas of Europe and have an important impact on livestock industry. More importanly, the cercariae of Orientobilharzia sp. can also infect human, in which they can cause cercarial dermatitis, so orientobilharziasis is an important zoonosis parasitic disease. Early accurate diagnosis for orientobilharziasis is an important means that control the morbidity and the disease to spread effectively. The traditional diagnosis method mainly is the etiology inspection and the immunology examination, such as IHA、ELISA and so on. However the cause of disease inspection detection rate is low, and the acute stage have processed, then, it has delayed the effective treatment opportunity, thereby, it affect the control of primary disease, the immunology examination commonly used antigen usually come from the worm and the egg, these two kinds of antigen ingredient is complex, the preparation cost is high, the period is long, so it limits application for this method extensively. Therefore, this experiment plans to use the molecular biology method, clone the 14-3-3 gene using RACE technology and prepares the reorganization protein, establishes the indirect ELISA diagnosis method.
Specific primers were designed and synthesized according to the reported 14-3-3 gene from Schistosoma japonicum and S. mansoni in GenBankTM. Intermediate fragment of 14-3-3 gene was amplified by RT-PCR, then cloned into pMD18-T vector and transformed into the competent Escherichia coli JM109 for obtaining the positive clone and sequenced. According to the sequenced information, another specific primers were designed again, the 3’end fragment and 5’end fragment of 14-3-3 gene were obtained by 3’RACE and 5’RACE. And then, these three fragment sequence were spliced by molecular biology software.
Then specific primers having restriction endonuclease digestion wites were designed and synthesized according to the total length of cDNA sequence in O. turkestanicum. The gene 14-3-3 was amplified by RT-PCR, then cloned into pMD18-T vector and transformed into the competent E. coli JM109 for obtaining the positive clone, the recombinant plasmid pMD18-T/14-3-3 was digested with EcoRⅠand XhoⅠ, and then sequenced. In order to express, the gene 14-3-3 was subcloned into pET-30a. The positive plasmid pMD18-T-14-3-3 and the expression vector pET-30a(+)were digested by restriction endonuclease digestion and reclamation, then transformed into the competent E. coli JM109, extracted the plasmid and identified, then the positive plasmid was transformed into the optimization competent E. coli BL21, hence, the recombinant strain E. coli BL21 (pET-30a-14-3-3) expressing fusion protein 14-3-3 was constructed. It was proved that the recombinant plasmid pET-30a-14-3-3 contained the fusion gene 14-3-3, which had correct sequence and ORF by identification of restriction endonuclease digestion and sequence analysis. The recombinant strain E. coli BL21 (pET-30a-14-3-3) was innoculated into LB medium (included Kan 50μg/mL) at the ratio of 1%, and cultured for 2.5 hours at 37℃, then induced by 1mmol/L IPTG for 4h, finally, the fusion protein 14-3-3 was expressed at high level. The expressed protein could be recognized by antibodies of O. turkestanicum by western blot.
The ELISA plate was coated by the purified recombinant 14-3-3 antigen, the reaction conditions of indirect ELISA were determined by experiment and optimization. The optimal coating antigen for microplate was 5.0μg/mL, and the optimal coating buffer was carbonate buffer solution (0.05M pH9.6). Five percent skim milk was added as the optimal blocking agent and incubated for 1 hour at 37℃. The optimal dilution of sera samples was 1:100, and the best serum dilution buffer was 5% skim milk. The working concentration of HRP-labeled rabbit anti-sheep IgG was 1:5 000. The cut off value for indirect ELISA was determined from 105 negative ovine serum samples by statistics analysis. If the OD value is more than or equal to 0.31, the test sample will be positive, or else negative. 65 positive and 105 negative serum samples from the sheep and goats (confirmed by slaughtered) were detected for the antibodies against O. turkestanicum with the developed indirect ELISA. It was found that the specificity and sensitivity of the developed indirect ELISA was 91.5% and 88.6% respectively. The indirect ELISA had no cross-reactivity with positive serum of Fasciola hepatica, Paramphistornum cervi, Monieziaexpansa and Haemonchus contortus, and so on. A total of 240 serum samples of the sheep and goats from Heilongjiang province were used to evaluate serological screening for orientobilharziasis by developed indirect ELISA. The result indicated that the seroprevalence of orientobilharziasis is 35.3%, and similar with the results of reality status. Key words: Orientobilharziasis, the protein 14-3-3, RACE, recombinant antigen, indirect ELISA
本试验根据GenBankTM上发表的日本血吸虫、曼氏血吸虫和牛血吸虫信号蛋白14-3-3序列,依其保守区设计合成一对引物,以提取的东毕吸虫总RNA为模板,PT-PCR方法扩增信号蛋白14-3-3基因的中间片段,连接pMD18-T载体并转化到TG1宿主菌中,经鉴定,获得阳性克隆,并进行序列分析。根据测得的序列信息,再设计一对引物,利用5’RACE和3’RACE技术分别扩增目的基因的5’端和3’端,鉴定正确后并测序。利用分子生物学软件拼接三段序列得到全长序列信息。
其次,根据东毕吸虫信号蛋白14-3-3全长cDNA序列设计两条加有EcoRⅠ、XhoⅠ酶切位点的引物,利用RT-PCR从东毕吸虫总RNA中扩增信号蛋白14-3-3全长序列,连接pMD18-T载体并转化到TG1宿主菌中,经鉴定及序列分析,获得阳性克隆。阳性质粒pMD18-T-信号蛋白14-3-3和表达载体pET-30a(+),经EcoRⅠ、XhoⅠ双酶切后回收、连接、转化到TG1宿主菌,提取质粒,酶切鉴定正确后,阳性质粒转化到最佳宿主菌BL21(DE3)中,构建其原核重组表达载体pET-30a-信号蛋白14-3-3,经酶切鉴定和DNA序列分析证实,构建的重组质粒pET-30a-信号蛋白14-3-3中含有信号蛋白14-3-3基因,且基因序列和阅读框架均正确。将重组菌株BL21(pET-30a-信号蛋白14-3-3)按1%的量接种到含有Kan(50μg/mL)的LB液体培养基中,37℃振荡培养2.5h,然后用1mmol/L IPTG诱导4h,融合目的蛋白获得高效表达。经Western blot检测,重组菌株表达的融合蛋白能够被东毕吸虫特异性抗体所识别。
以纯化的重组信号蛋白14-3-3作为抗原包被酶标板,通过对间接ELISA各反应条件的优化,确定了最佳反应条件为:最佳封闭液为5%脱脂奶粉,封闭条件为37℃1h;抗原最佳包被浓度为5.0μg/mL;抗原的最佳包被液为50mM pH9.6的碳酸盐缓冲液;最佳血清稀释液为5%脱脂奶粉,血清稀释倍数为100倍;HRP-兔抗羊IgG的最适工作浓度为1:5 000。采用已确立的间接ELISA反应条件,对105份阴性血清的检测结果进行统计学分析,确定了间接ELISA方法判定标准,即OD450值大于等于0.31为阳性,小于0.31为阴性。应用建立的间接ELISA方法对105份阴性血清和65份阳性血清(经剖检法和形态鉴定证实)的检测结果表明,ELISA方法的特异性为91.5%,敏感性为88.6%。而且与肝片吸虫、鹿前后盘吸虫、扩展莫尼茨绦虫和捻转血矛线虫阳性血清均无交叉反应。采用本研究建立的方法,对来自于不同地区的240份绵羊血清进行了检测,结果阳性率为35.3%,与省内的感染情况基本相似。
Adult worms of Orientobilharzia turkestanicum live in the portal veins or intestinal veins of cattle, sheep and other animals and cause orientobilharziasis. It is widely distributed in Asia and several areas of Europe and have an important impact on livestock industry. More importanly, the cercariae of Orientobilharzia sp. can also infect human, in which they can cause cercarial dermatitis, so orientobilharziasis is an important zoonosis parasitic disease. Early accurate diagnosis for orientobilharziasis is an important means that control the morbidity and the disease to spread effectively. The traditional diagnosis method mainly is the etiology inspection and the immunology examination, such as IHA、ELISA and so on. However the cause of disease inspection detection rate is low, and the acute stage have processed, then, it has delayed the effective treatment opportunity, thereby, it affect the control of primary disease, the immunology examination commonly used antigen usually come from the worm and the egg, these two kinds of antigen ingredient is complex, the preparation cost is high, the period is long, so it limits application for this method extensively. Therefore, this experiment plans to use the molecular biology method, clone the 14-3-3 gene using RACE technology and prepares the reorganization protein, establishes the indirect ELISA diagnosis method.
Specific primers were designed and synthesized according to the reported 14-3-3 gene from Schistosoma japonicum and S. mansoni in GenBankTM. Intermediate fragment of 14-3-3 gene was amplified by RT-PCR, then cloned into pMD18-T vector and transformed into the competent Escherichia coli JM109 for obtaining the positive clone and sequenced. According to the sequenced information, another specific primers were designed again, the 3’end fragment and 5’end fragment of 14-3-3 gene were obtained by 3’RACE and 5’RACE. And then, these three fragment sequence were spliced by molecular biology software.
Then specific primers having restriction endonuclease digestion wites were designed and synthesized according to the total length of cDNA sequence in O. turkestanicum. The gene 14-3-3 was amplified by RT-PCR, then cloned into pMD18-T vector and transformed into the competent E. coli JM109 for obtaining the positive clone, the recombinant plasmid pMD18-T/14-3-3 was digested with EcoRⅠand XhoⅠ, and then sequenced. In order to express, the gene 14-3-3 was subcloned into pET-30a. The positive plasmid pMD18-T-14-3-3 and the expression vector pET-30a(+)were digested by restriction endonuclease digestion and reclamation, then transformed into the competent E. coli JM109, extracted the plasmid and identified, then the positive plasmid was transformed into the optimization competent E. coli BL21, hence, the recombinant strain E. coli BL21 (pET-30a-14-3-3) expressing fusion protein 14-3-3 was constructed. It was proved that the recombinant plasmid pET-30a-14-3-3 contained the fusion gene 14-3-3, which had correct sequence and ORF by identification of restriction endonuclease digestion and sequence analysis. The recombinant strain E. coli BL21 (pET-30a-14-3-3) was innoculated into LB medium (included Kan 50μg/mL) at the ratio of 1%, and cultured for 2.5 hours at 37℃, then induced by 1mmol/L IPTG for 4h, finally, the fusion protein 14-3-3 was expressed at high level. The expressed protein could be recognized by antibodies of O. turkestanicum by western blot.
The ELISA plate was coated by the purified recombinant 14-3-3 antigen, the reaction conditions of indirect ELISA were determined by experiment and optimization. The optimal coating antigen for microplate was 5.0μg/mL, and the optimal coating buffer was carbonate buffer solution (0.05M pH9.6). Five percent skim milk was added as the optimal blocking agent and incubated for 1 hour at 37℃. The optimal dilution of sera samples was 1:100, and the best serum dilution buffer was 5% skim milk. The working concentration of HRP-labeled rabbit anti-sheep IgG was 1:5 000. The cut off value for indirect ELISA was determined from 105 negative ovine serum samples by statistics analysis. If the OD value is more than or equal to 0.31, the test sample will be positive, or else negative. 65 positive and 105 negative serum samples from the sheep and goats (confirmed by slaughtered) were detected for the antibodies against O. turkestanicum with the developed indirect ELISA. It was found that the specificity and sensitivity of the developed indirect ELISA was 91.5% and 88.6% respectively. The indirect ELISA had no cross-reactivity with positive serum of Fasciola hepatica, Paramphistornum cervi, Monieziaexpansa and Haemonchus contortus, and so on. A total of 240 serum samples of the sheep and goats from Heilongjiang province were used to evaluate serological screening for orientobilharziasis by developed indirect ELISA. The result indicated that the seroprevalence of orientobilharziasis is 35.3%, and similar with the results of reality status. Key words: Orientobilharziasis, the protein 14-3-3, RACE, recombinant antigen, indirect ELISA
引文
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