东方田鼠日本血吸虫天然抗性相关基因的筛选和验证
发布时间：2018-08-01 17:33
【摘要】： 日本血吸虫病(schistosomiasis japonica)作为人兽共患寄生虫病,在我国仍然是一个重要的公共卫生问题。现阶段我国实行的是“以控制传染源”为主、综合治理的血吸虫病防治策略。其中,吡喹酮群体化疗是目前血吸虫病防治措施的基础,但血吸虫病仍在向新的地区蔓延,并且吡喹酮化疗也有一定的局限性,群体化疗并不能防止重复感染,还可能会产生吡喹酮抗性株。因此,在血吸虫病防治策略中,疫苗策略已被认为将是吡喹酮化疗措施的重要补充。目前已有酶性、肌性、膜相关性等多种疫苗候选抗原的基因得到了克隆和表达,并且开展了动物保护性实验,虽然取得了一定的预期效果,但继续寻找新的候选抗原分子以及提高候选疫苗分子的免疫原性仍是血吸虫病疫苗研究的重要方向,通过免疫筛选cDNA文库去发掘更有效的疫苗候选分子是获得新的候选抗原分子的有效途径。 发展疫苗的主要目的是降低虫荷和虫卵在肝组织的沉积,因此疫苗的效应应多针对童虫阶段和成虫产卵。东方田鼠(Microtus fortis,Mf)是一种感染日本血吸虫后不致病的哺乳类动物。血吸虫童虫可能是宿主免疫系统较为适合的靶子,若能找到童虫阶段的特异性抗原分子,将血吸虫消灭在此阶段或阻止其生长、发育、成熟、产卵和致病,这不仅可以减轻血吸虫病所造成的病理损害,还可有效地阻止其传播。因此用东方田鼠血清免疫筛选日本血吸虫童虫cDNA文库中,以寻找相关的疫苗候选分子,可能会获得令人满意的结果,并为东方田鼠天然抗日本血吸虫机制的研究提供信息。 精氨酸甲基化在血吸虫基因表达调节中起着重要的作用。这是一种翻译后修饰,它参与了多种细胞功能,包括RNA加工处理、细胞信号转导、蛋白亚细胞定位、转录后调节和DNA修复。 高迁移率族蛋白B1(HMGB1)参与基因转录、复制、重组与修复。胞外HMGB1是一种重要的晚期炎症介质,它可以激活巨噬细胞释放TNF-α和IL-13等早期炎症因子。在血吸虫感染中,TNF-α和IL-13对虫卵周围肉芽肿的形成起着重要的免疫诱导作用,可能是宿主感染后免疫调节的关键分子。HMGB1与一些感染性疾病的发病密切相关。 本研究从日本血吸虫童虫cDNA文库筛选得到的阳性克隆中选择了蛋白质精氨酸甲基转移酶1(PRMT1)和高迁移率族蛋白B1(HMGB1)编码基因进行研究。首先通过生物信息学分析获得其完整的开放阅读框,然后通过分子克隆技术对这2个基因进行了克隆表达。随后,对纯化重组蛋白reSjcHMGB1开展了动物免疫保护性试验,评价其作为疫苗候选抗原的价值。 一、东方田鼠日本血吸虫天然抗性相关基因的免疫筛选 用日本血吸虫天然抗性东方田鼠血清免疫筛选日本血吸虫肝期童虫cDNA文库,将3次复筛获得的阳性克隆转入大肠杆菌(E.coil)BM25.8环化成质粒,抽提质粒DNA,EcoRⅠ和HindⅢ双酶切琼脂糖凝胶电泳鉴定,插入片段进行核苷酸序列测序,并进行生物信息学分析。结果,经3次复筛后获得32个阳性克隆,插入片段为300 bp～1 100 bp之间,测序结果经同源性分析,共获得26个不同分子基因:高迁移率族蛋白B1(HMGB1)部分基因,蛋白质精氨酸甲基转移酶部分编码基因,细胞色素b部分编码基因,线粒体编码区基因,16个日本血吸虫未知蛋白编码基因,6个日本血吸虫未知新基因。本研究用东方田鼠血清筛选日本血吸虫童虫cDNA文库,获得一批新的日本血吸虫疫苗候选分子的编码基因,为研究血吸虫病疫苗和血吸虫病免疫诊断奠定了基础。 二、日本血吸虫蛋白质精氨酸甲基转移酶(PRMT)1编码基因的克隆、表达和分析 依据电子延伸得到的SjPRMT1基因序列设计一对引物,上游引物引入BamHⅠ酶切位点,下游引物引入XhoⅠ酶切位点。以日本血吸虫成虫总RNA为模板,经反转录PCR(RT-PCR)扩增目的编码基因。纯化PCR产物与pGEM-T载体连接后转化感受态大肠杆菌JM109,抽提重组质粒DNA用BamHⅠ和XhoⅠ双酶切及核苷酸序列测序进行鉴定。选择阅读框正确的克隆,纯化重组质粒中目的基因双酶切片段,亚克隆入pET28a原核表达载体,构建重组质粒pET28a-SjPRMT1,转化DH5α感受态菌,重组质粒经双酶切和核苷酸序列鉴定后,阳性克隆质粒转化感受态大肠杆菌BL21(DE3),IPTG诱导表达并获得纯化的重组蛋白(简称为reSjPRMT1),采用SDS-PAGE和Western blotting分析和鉴定该重组蛋白。运用Gene Runner软件预测reSjPRMT1蛋白的二级结构、功能位点及表位特征。结果,RT-PCR扩增出一大小与预期一致的基因片段。TA克隆插入目的片段经核苷酸序列测定,cDNA全长1083 bp,编码360个氨基酸。序列分析表明该片段与SmPRMT1基因序列同源性为87%,推导的氨基酸序列同源性为95%。表达蛋白经SDS-PAGE和Western blotting分析显示,reSjPRMT1重组蛋白的分子质量约43 kDa(包括6个组氨酸),以可溶性方式表达,可被日本血吸虫感染小鼠血清和抗His-G HRP抗体识别。SjPRMT1基因的克隆、表达获得成功,并获得纯化的重组蛋白,为今后进一步研究其生物学特性以及免疫原性奠定了基础。 三、日本血吸虫高迁移率族蛋白B1(HMGB1)编码基因的克隆、表达和免疫保护性研究 依据公布的SmHMGB1基因序列设计一对简并引物,上游引物引入BamHⅠ酶切位点,下游引物引入SalⅠ酶切位点。以日本血吸虫成虫总RNA为模板,经反转录PCR(RT-PCR)扩增目的编码基因。纯化PCR产物与pGEM-T载体连接后转化感受态大肠杆菌JM109,抽提重组质粒DNA用BamHⅠ和SalⅠ双酶切及核苷酸序列测序进行鉴定。选择阅读框正确的克隆,纯化重组质粒中目的基因双酶切片段,亚克隆入pET28a原核表达载体,构建重组质粒pET28a-SjHMGB1,转化DH5α感受态菌,重组质粒经双酶切和核苷酸序列鉴定后,阳性克隆质粒转化感受态大肠杆菌BL21(DE3),IPTG诱导表达并获得纯化的重组蛋白,采用SDS-PAGE和Western blotting分析和鉴定该重组蛋白。运用Gene Runner软件预测reSjHMGB1的二级结构、功能位点及表位特征。在免疫保护性实验中,雌性C57BL/6小鼠随机分为5组,分别为感染对照组、弗氏佐剂对照组、MontanideISA206佐剂对照组、reSjcHMGB1加弗氏佐剂免疫组、reSicHMGB1加MontanideISA 206佐剂免疫组。感染对照组不注射任何抗原和佐剂,两种佐剂对照组小鼠注射乳化的生理盐水加弗氏或Montanide ISA 206佐剂,两免疫组每只小鼠经背部皮下多点注射乳化的20μg reSjcHMGB1加弗氏或Montanide ISA 206佐剂,共免疫3次,间隔2周。末次免疫后2周,小鼠经腹部感染日本血吸虫尾蚴30±1条,攻击感染后6周剖杀小鼠,进行成虫和虫卵计数。并分别于免疫前、攻击感染前和小鼠剖杀前采血并分离血清,ELISA检测血清中特异性IgG抗体。结果,RT-PCR扩增的目的片段经琼脂糖凝胶电泳观察,与预计的一致。TA克隆插入目的片段经核苷酸序列测定,cDNA全长531 bp,编码176个氨基酸。序列分析表明该片段与SmHMGB1基因序列同源性为86%,推导的氨基酸序列同源性为93%。表达蛋白经SDS-PAGE分析显示,reSjHMG重组蛋白的分子质量约30 kDa(包括6个组氨酸),以可溶性方式表达。免疫印迹结果显示,日本血吸虫感染小鼠血清、重组抗原免疫小鼠血清和抗His-G HRP抗体均可识别该重组蛋白。生物信息学分析表明该蛋白包含两个保守的结构域(A盒和B盒)及含酸性氨基酸的C末端,同时存在多个潜在的抗原决定簇。在免疫保护性实验中,ELISA结果表明,免疫后重组抗原加两种佐剂免疫组小鼠的特异性IgG抗体水平均显著高于感染对照组和佐剂对照组(P＜0.05)。reSjcHMGB1加弗氏佐剂免疫组减虫率和肝组织减卵率分别为17.9%和17.6%,其虫荷数和每克肝组织虫卵数(EPG)与感染对照组相比均无统计学意义(P＞0.05)。reSjcHMGB1加Montanide ISA 206佐剂免疫组减虫率和肝组织减卵率分别为分别为33.2%和11.3%,其虫荷数与感染对照组相比有统计学意义(P＜0.05),但与ISA 206佐剂对照组相比无统计学意义(P＞0.05)。本研究成功克隆、表达SjHMGB1,并获得纯化的重组蛋白。在动物保护性实验中,重组抗原并未诱导小鼠产生明显的抗感染和抗生殖免疫保护作用。
[Abstract]:Schistosomiasis japonica, as a zoonotic parasitic disease, is still an important public health problem in China. At the present stage, the prevention and control strategy of schistosomiasis, which is based on the control of the source of infection and comprehensive treatment, is the basis for the prevention and treatment of schistosomiasis, but the treatment of the disease is the basis of the prevention and treatment of schistosomiasis. Schistosomiasis is still spreading to the new area, and the chemotherapy of praziquantel has certain limitations. Group chemotherapy does not prevent repeated infection and may produce the resistance strain of the praziquantel. Therefore, in the strategy of schistosomiasis control, the vaccine strategy has been considered to be an important supplement to the praziquantel treatment. The genes of a variety of vaccine candidate antigens have been cloned and expressed, and animal protection experiments have been carried out. Although a certain expected effect has been achieved, it is still an important direction to continue to search for new candidate antigens and improve the immunogenicity of the candidate vaccine molecules, and the cDNA text is screened by immunization. To find more effective vaccine candidates is an effective way to obtain new candidate antigen molecules.
The main purpose of the development of the vaccine is to reduce the deposition of worm and egg in the liver tissue, so the effect of the vaccine should be more aimed at the spawning of the adult and the adult. Microtus Fortis (Mf) is a mammalian animal that is not pathogenic after infection of Schistosoma japonicum. To find out the specific antigen molecules in the stage of the child's insect, eliminate the schistosomiasis at this stage or prevent its growth, development, maturation, spawning and pathogenicity, which can not only reduce the pathological damage caused by schistosomiasis, but also effectively prevent its transmission. Therefore, the cDNA Library of Schistosoma japonicum is screened by the immunization of the serum of voles orient in order to find the phase. These results may provide information for the study of the natural mechanism of resistance of Microtus Fortis to Schistosoma japonicum.
Arginine methylation plays an important role in the regulation of gene expression in Schistosoma. This is a post-translational modification that participates in a variety of cell functions, including RNA processing, cell signal transduction, protein subcellular localization, post transcriptional regulation and DNA repair.
High mobility group protein B1 (HMGB1) participates in gene transcription, replication, recombination and repair. Extracellular HMGB1 is an important late inflammatory mediator, which activates the release of early inflammatory factors such as TNF- A and IL-13 in macrophages. In Schistosoma infection, TNF- alpha and IL-13 play an important role in inducing the formation of buds around the eggs. .HMGB1, a key molecule of host immune regulation after infection, is closely related to the pathogenesis of some infectious diseases.
In this study, the positive clones screened from the cDNA Library of Schistosoma japonicum were selected to study the protein arginine methyltransferase 1 (PRMT1) and high mobility group protein B1 (HMGB1). First, the complete open reading frame was obtained by bioinformatics analysis, and then the 2 genes were introduced by molecular cloning technology. Subsequently, the purified recombinant protein reSjcHMGB1 was cloned and expressed in vivo, and its value as a vaccine candidate antigen was evaluated.
Immunological screening of genes related to natural resistance of Schistosoma japonicum in Microtus fortis
The cDNA Library of Schistosoma japonicum liver stage was screened using the natural resistance of Schistosoma japonicum resistance to the serum of voles Orient. The positive clones obtained from 3 times of rescreening were transformed into Escherichia coli (E.coil) BM25.8 cyclization plasmids, DNA, EcoR I and Hind III double enzyme cut agarose gel electrophoresis, and the inserted fragment was sequenced. Bioinformatics analysis. Results, 32 positive clones were obtained after 3 rescreening. The inserted fragments were 300 BP to 1100 BP. The sequencing results obtained 26 different molecular genes: high mobility group protein B1 (HMGB1) partial gene, protein arginine methyltransferase partial encoding gene, and cytochrome b encoding gene, Mitochondrial coding region gene, 16 unknown protein encoding gene of Schistosoma japonicum, and 6 unknown new genes of Schistosoma japonicum. This study uses the serum of vole vole to screen the cDNA Library of Schistosoma japonicum, and obtains a number of new coding genes for the candidate of Schistosoma japonicum vaccine, which lays a foundation for the study of schistosomiasis vaccine and schistosomiasis immunity diagnosis. Set the foundation.
Two, cloning, expression and analysis of the gene encoding protein arginine methyltransferase (PRMT) 1 of Schistosoma japonicum.
A pair of primers was designed based on the SjPRMT1 gene sequence obtained by the electron extension. The upstream primers introduced the BamH I enzyme cut site and the downstream primers introduced the Xho I enzyme cut site. The target encoding gene of the total RNA of Schistosoma japonicum was amplified by reverse transcriptional PCR (RT-PCR). The purified PCR product was connected with pGEM-T vector to convert the susceptible Escherichia coli JM109. The recombinant plasmid DNA was identified by BamH I and Xho I double enzyme digestion and nucleotide sequence sequencing. Select the correct clone of the reading frame, purify the double enzyme cut fragment of the target gene in the recombinant plasmid, subclone into the pET28a prokaryotic expression vector, construct the recombinant plasmid pET28a-SjPRMT1, transform the DH5 alpha receptive bacteria, and the recombinant plasmid through double enzyme cutting and nucleotides. After the sequence identification, the positive cloned plasmid transformed the receptive Escherichia coli BL21 (DE3), IPTG induced the expression and obtained the purified recombinant protein (referred to as reSjPRMT1). The recombinant protein was analyzed and identified by SDS-PAGE and Western blotting. The Gene Runner software was used to predict the two structure of the reSjPRMT1 protein, the functional site and the epitope characteristics. Results, RT. -PCR amplified a size and expected gene fragment.TA clones to be inserted into the target fragment by nucleotide sequence and cDNA was 1083 BP and encoded 360 amino acids. Sequence analysis showed that the fragment was homologous with the SmPRMT1 gene sequence of 87%. The deduced amino acid sequence homology was analyzed by SDS-PAGE and Western blotting analysis of 95%. expression protein. The results showed that the molecular mass of the recombinant protein of reSjPRMT1 was about 43 kDa (including 6 histidine), expressed in a soluble way, could be cloned by the sera of mice infected with Schistosoma japonicum and the anti His-G HRP antibody to identify the.SjPRMT1 gene. The expression was successful, and the purified recombinant protein was obtained. It could further study its biological characteristics and immunogenicity for the future. The foundation is laid.
Three, cloning, expression and immunological protection of high mobility group protein B1 (HMGB1) gene of Schistosoma japonicum.
A pair of degenerate primers was designed according to the published SmHMGB1 gene sequence. The upstream primers introduced the BamH I enzyme cutting site and the downstream primers introduced the Sal I enzyme cut site. The total RNA of the adult Schistosoma japonicum was used as the template, and the target encoding gene was amplified by the reverse transcription PCR (RT-PCR). The purified PCR product was connected with pGEM-T vector to convert the susceptible Escherichia coli JM109 and pumped. The recombinant plasmid DNA was identified by BamH I and Sal I double enzyme digestion and nucleotide sequence sequencing. Select the correct clone of the reading frame, purify the double enzyme cut fragment of the target gene in the recombinant plasmid, subclone into the pET28a prokaryotic expression vector, construct the recombinant plasmid pET28a-SjHMGB1, transform the DH5 alpha susceptible strain, and the recombinant plasmid through double enzyme cutting and nucleotide sequence. After identification, the positive cloned plasmid transformed the receptive Escherichia coli BL21 (DE3), IPTG induced expression and obtained the purified recombinant protein. The recombinant protein was analyzed and identified by SDS-PAGE and Western blotting. The Gene Runner software was used to predict the two stage structure of reSjHMGB1, the functional loci and the epitope characteristics. In the immunoprotective experiment, female C57BL/6. Mice were randomly divided into 5 groups: infection control group, Freund's adjuvant control group, MontanideISA206 adjuvant control group, reSjcHMGB1 Freund adjuvant immune group, reSicHMGB1 plus MontanideISA 206 adjuvant immune group. The infection control group was not injected with any antigen and adjuvant, two kinds of adjuvant injected phacoemulsification saline or Montan to mice. IDE ISA 206 adjuvant, two immunization groups were injected into the back subcutaneous injection of 20 mu g reSjcHMGB1 and Montanide ISA 206 adjuvant, immunized for 3 times, interval 2 weeks. After the last immunization, the mice infected with Schistosoma japonicum cercariae 30 + 1 weeks after the last immunization. The mice were killed 6 weeks after the infection, and the count of adults and eggs was carried out. And respectively. Before the immunization, the blood was collected before and before the infection of the infected mice and the serum was isolated, and the specific IgG antibody in the serum was detected by ELISA. Results the target fragment of RT-PCR amplification was observed by agarose gel electrophoresis, and the same.TA clone inserted into the target fragment was determined by nucleotide sequence. The total length of cDNA was 531 BP and encoded 176 amino acids. Sequence analysis showed that this The homology of the fragment and SmHMGB1 gene sequence was 86%. The deduced amino acid sequence homology was 93%. expression protein by SDS-PAGE analysis. The molecular weight of the recombinant protein of reSjHMG was about 30 kDa (including 6 histidine) and expressed in a soluble way. The result of immunoblotting showed that the mice sera infected by Schistosoma japonicum and the recombinant antigen were immune to the mice serum. And anti His-G HRP antibodies can identify the recombinant protein. Bioinformatics analysis shows that the protein contains two conservative domains (A box and B box) and the C terminal of acid amino acids, and there are several potential antigenic determinants. In the immunoprotective experiment, ELISA results show that the post immune recombinant antigen and two adjuvant immune groups are small. The level of specific IgG antibody in rats was significantly higher than that in the infection control group and the adjuvant control group (P < 0.05).ReSjcHMGB1 gf's adjuvant immune group and the liver tissue reduction rate were 17.9% and 17.6% respectively. The number of worms and the number of eggs per gram of liver tissue (EPG) were not statistically significant (P > 0.05).ReSjcHMGB1 plus Montanide (P > 0.05) and Montanide The rate of worm reduction and liver tissue reduction were 33.2% and 11.3% respectively in the ISA 206 adjuvant immunization group, respectively, and the number of parasite was statistically significant compared with the control group (P < 0.05), but it was not statistically significant compared with the ISA 206 adjuvant control group (P > 0.05). This study was successfully cloned, expressed SjHMGB1, and obtained the purified recombinant protein. In the experiment, recombinant antigen did not induce significant anti infection and anti reproductive immunity in mice.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383.2
本文编号：2158321
[Abstract]:Schistosomiasis japonica, as a zoonotic parasitic disease, is still an important public health problem in China. At the present stage, the prevention and control strategy of schistosomiasis, which is based on the control of the source of infection and comprehensive treatment, is the basis for the prevention and treatment of schistosomiasis, but the treatment of the disease is the basis of the prevention and treatment of schistosomiasis. Schistosomiasis is still spreading to the new area, and the chemotherapy of praziquantel has certain limitations. Group chemotherapy does not prevent repeated infection and may produce the resistance strain of the praziquantel. Therefore, in the strategy of schistosomiasis control, the vaccine strategy has been considered to be an important supplement to the praziquantel treatment. The genes of a variety of vaccine candidate antigens have been cloned and expressed, and animal protection experiments have been carried out. Although a certain expected effect has been achieved, it is still an important direction to continue to search for new candidate antigens and improve the immunogenicity of the candidate vaccine molecules, and the cDNA text is screened by immunization. To find more effective vaccine candidates is an effective way to obtain new candidate antigen molecules.
The main purpose of the development of the vaccine is to reduce the deposition of worm and egg in the liver tissue, so the effect of the vaccine should be more aimed at the spawning of the adult and the adult. Microtus Fortis (Mf) is a mammalian animal that is not pathogenic after infection of Schistosoma japonicum. To find out the specific antigen molecules in the stage of the child's insect, eliminate the schistosomiasis at this stage or prevent its growth, development, maturation, spawning and pathogenicity, which can not only reduce the pathological damage caused by schistosomiasis, but also effectively prevent its transmission. Therefore, the cDNA Library of Schistosoma japonicum is screened by the immunization of the serum of voles orient in order to find the phase. These results may provide information for the study of the natural mechanism of resistance of Microtus Fortis to Schistosoma japonicum.
Arginine methylation plays an important role in the regulation of gene expression in Schistosoma. This is a post-translational modification that participates in a variety of cell functions, including RNA processing, cell signal transduction, protein subcellular localization, post transcriptional regulation and DNA repair.
High mobility group protein B1 (HMGB1) participates in gene transcription, replication, recombination and repair. Extracellular HMGB1 is an important late inflammatory mediator, which activates the release of early inflammatory factors such as TNF- A and IL-13 in macrophages. In Schistosoma infection, TNF- alpha and IL-13 play an important role in inducing the formation of buds around the eggs. .HMGB1, a key molecule of host immune regulation after infection, is closely related to the pathogenesis of some infectious diseases.
In this study, the positive clones screened from the cDNA Library of Schistosoma japonicum were selected to study the protein arginine methyltransferase 1 (PRMT1) and high mobility group protein B1 (HMGB1). First, the complete open reading frame was obtained by bioinformatics analysis, and then the 2 genes were introduced by molecular cloning technology. Subsequently, the purified recombinant protein reSjcHMGB1 was cloned and expressed in vivo, and its value as a vaccine candidate antigen was evaluated.
Immunological screening of genes related to natural resistance of Schistosoma japonicum in Microtus fortis
The cDNA Library of Schistosoma japonicum liver stage was screened using the natural resistance of Schistosoma japonicum resistance to the serum of voles Orient. The positive clones obtained from 3 times of rescreening were transformed into Escherichia coli (E.coil) BM25.8 cyclization plasmids, DNA, EcoR I and Hind III double enzyme cut agarose gel electrophoresis, and the inserted fragment was sequenced. Bioinformatics analysis. Results, 32 positive clones were obtained after 3 rescreening. The inserted fragments were 300 BP to 1100 BP. The sequencing results obtained 26 different molecular genes: high mobility group protein B1 (HMGB1) partial gene, protein arginine methyltransferase partial encoding gene, and cytochrome b encoding gene, Mitochondrial coding region gene, 16 unknown protein encoding gene of Schistosoma japonicum, and 6 unknown new genes of Schistosoma japonicum. This study uses the serum of vole vole to screen the cDNA Library of Schistosoma japonicum, and obtains a number of new coding genes for the candidate of Schistosoma japonicum vaccine, which lays a foundation for the study of schistosomiasis vaccine and schistosomiasis immunity diagnosis. Set the foundation.
Two, cloning, expression and analysis of the gene encoding protein arginine methyltransferase (PRMT) 1 of Schistosoma japonicum.
A pair of primers was designed based on the SjPRMT1 gene sequence obtained by the electron extension. The upstream primers introduced the BamH I enzyme cut site and the downstream primers introduced the Xho I enzyme cut site. The target encoding gene of the total RNA of Schistosoma japonicum was amplified by reverse transcriptional PCR (RT-PCR). The purified PCR product was connected with pGEM-T vector to convert the susceptible Escherichia coli JM109. The recombinant plasmid DNA was identified by BamH I and Xho I double enzyme digestion and nucleotide sequence sequencing. Select the correct clone of the reading frame, purify the double enzyme cut fragment of the target gene in the recombinant plasmid, subclone into the pET28a prokaryotic expression vector, construct the recombinant plasmid pET28a-SjPRMT1, transform the DH5 alpha receptive bacteria, and the recombinant plasmid through double enzyme cutting and nucleotides. After the sequence identification, the positive cloned plasmid transformed the receptive Escherichia coli BL21 (DE3), IPTG induced the expression and obtained the purified recombinant protein (referred to as reSjPRMT1). The recombinant protein was analyzed and identified by SDS-PAGE and Western blotting. The Gene Runner software was used to predict the two structure of the reSjPRMT1 protein, the functional site and the epitope characteristics. Results, RT. -PCR amplified a size and expected gene fragment.TA clones to be inserted into the target fragment by nucleotide sequence and cDNA was 1083 BP and encoded 360 amino acids. Sequence analysis showed that the fragment was homologous with the SmPRMT1 gene sequence of 87%. The deduced amino acid sequence homology was analyzed by SDS-PAGE and Western blotting analysis of 95%. expression protein. The results showed that the molecular mass of the recombinant protein of reSjPRMT1 was about 43 kDa (including 6 histidine), expressed in a soluble way, could be cloned by the sera of mice infected with Schistosoma japonicum and the anti His-G HRP antibody to identify the.SjPRMT1 gene. The expression was successful, and the purified recombinant protein was obtained. It could further study its biological characteristics and immunogenicity for the future. The foundation is laid.
Three, cloning, expression and immunological protection of high mobility group protein B1 (HMGB1) gene of Schistosoma japonicum.
A pair of degenerate primers was designed according to the published SmHMGB1 gene sequence. The upstream primers introduced the BamH I enzyme cutting site and the downstream primers introduced the Sal I enzyme cut site. The total RNA of the adult Schistosoma japonicum was used as the template, and the target encoding gene was amplified by the reverse transcription PCR (RT-PCR). The purified PCR product was connected with pGEM-T vector to convert the susceptible Escherichia coli JM109 and pumped. The recombinant plasmid DNA was identified by BamH I and Sal I double enzyme digestion and nucleotide sequence sequencing. Select the correct clone of the reading frame, purify the double enzyme cut fragment of the target gene in the recombinant plasmid, subclone into the pET28a prokaryotic expression vector, construct the recombinant plasmid pET28a-SjHMGB1, transform the DH5 alpha susceptible strain, and the recombinant plasmid through double enzyme cutting and nucleotide sequence. After identification, the positive cloned plasmid transformed the receptive Escherichia coli BL21 (DE3), IPTG induced expression and obtained the purified recombinant protein. The recombinant protein was analyzed and identified by SDS-PAGE and Western blotting. The Gene Runner software was used to predict the two stage structure of reSjHMGB1, the functional loci and the epitope characteristics. In the immunoprotective experiment, female C57BL/6. Mice were randomly divided into 5 groups: infection control group, Freund's adjuvant control group, MontanideISA206 adjuvant control group, reSjcHMGB1 Freund adjuvant immune group, reSicHMGB1 plus MontanideISA 206 adjuvant immune group. The infection control group was not injected with any antigen and adjuvant, two kinds of adjuvant injected phacoemulsification saline or Montan to mice. IDE ISA 206 adjuvant, two immunization groups were injected into the back subcutaneous injection of 20 mu g reSjcHMGB1 and Montanide ISA 206 adjuvant, immunized for 3 times, interval 2 weeks. After the last immunization, the mice infected with Schistosoma japonicum cercariae 30 + 1 weeks after the last immunization. The mice were killed 6 weeks after the infection, and the count of adults and eggs was carried out. And respectively. Before the immunization, the blood was collected before and before the infection of the infected mice and the serum was isolated, and the specific IgG antibody in the serum was detected by ELISA. Results the target fragment of RT-PCR amplification was observed by agarose gel electrophoresis, and the same.TA clone inserted into the target fragment was determined by nucleotide sequence. The total length of cDNA was 531 BP and encoded 176 amino acids. Sequence analysis showed that this The homology of the fragment and SmHMGB1 gene sequence was 86%. The deduced amino acid sequence homology was 93%. expression protein by SDS-PAGE analysis. The molecular weight of the recombinant protein of reSjHMG was about 30 kDa (including 6 histidine) and expressed in a soluble way. The result of immunoblotting showed that the mice sera infected by Schistosoma japonicum and the recombinant antigen were immune to the mice serum. And anti His-G HRP antibodies can identify the recombinant protein. Bioinformatics analysis shows that the protein contains two conservative domains (A box and B box) and the C terminal of acid amino acids, and there are several potential antigenic determinants. In the immunoprotective experiment, ELISA results show that the post immune recombinant antigen and two adjuvant immune groups are small. The level of specific IgG antibody in rats was significantly higher than that in the infection control group and the adjuvant control group (P < 0.05).ReSjcHMGB1 gf's adjuvant immune group and the liver tissue reduction rate were 17.9% and 17.6% respectively. The number of worms and the number of eggs per gram of liver tissue (EPG) were not statistically significant (P > 0.05).ReSjcHMGB1 plus Montanide (P > 0.05) and Montanide The rate of worm reduction and liver tissue reduction were 33.2% and 11.3% respectively in the ISA 206 adjuvant immunization group, respectively, and the number of parasite was statistically significant compared with the control group (P < 0.05), but it was not statistically significant compared with the ISA 206 adjuvant control group (P > 0.05). This study was successfully cloned, expressed SjHMGB1, and obtained the purified recombinant protein. In the experiment, recombinant antigen did not induce significant anti infection and anti reproductive immunity in mice.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383.2
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