【作者】 韩凯凯；

【导师】 李祥瑞；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2011， 博士

【摘要】 捻转血矛线虫(Haemonchus contortus)属毛圆科(Trichostrongylidae)血矛属(Haemonchus)线虫,分布较广,寄生于反刍动物第四胃,偶见于小肠,因吸血常常导致贫血、消瘦、严重时甚至死亡,对畜牧业造成重大经济损失。目前防治该病方法主要是化学药物驱虫,但由于虫体抗药性的产生以及药物残留和药物污染等问题,迫使人们将注意力转移到利用免疫学方法来防治该病。本文进行了捻转血矛线虫甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, HcGAPDH)特性研究及其疫苗免疫保护试验；同时对捻转血矛线虫烯醇化酶(Enolase, HcENO)进行了生物学特性研究,克隆得到捻转血矛线虫磷酸甘油酸激酶基因序列并对其进行了生物信息学分析,主要工作如下：1.捻转血矛线虫甘油醛-3-磷酸脱氢酶特性的研究根据GenBank上登录的捻转血矛线虫甘油醛-3-磷酸脱氢酶基因EST序列(登录号为AW670737,登录长度为362bp)设计基因特异性引物,分别利用5’RACE和3’RACE获得该基因5’端未知区和3’端未知区,从而拼接得到HcGAPDH基因的全长cDNA,然后将该序列递交GenBank(登录号为HM145749)。后经分析发现：该基因全长1303bp,含有最大开放阅读框(ORF)为1026bp,5’非翻译区(5’-UTR)长90 bp,3’非翻译区(3’-UTR)长175bp。根据此基因的全长cDNA设计1对特异性引物,以捻转血矛线虫的总RNA为模板,利用RT-PCR技术,扩增出约1000bp的产物,测序结果与推导出的ORF序列一致。BLAST分析后将该片段克隆到pET-28a(+)载体中,转化入感受态大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白。SDS-PAGE结果分析发现融合蛋白在上清和包涵体中均有表达,分子量约38kDa。以自然感染的山羊血清作为一抗,Western blot分析结果显示在38kDa处出现特异性条带。用HcGAPDH表达蛋白的包涵体免疫大鼠制备高免血清做一抗,Western blot分析HcGAPDH基因在捻转血矛线虫的天然表达,结果在38kD处有明显条带,与推测ORF的蛋白大小无明显区别。酶活性测定结果表明该重组蛋白具有一定的甘油醛-3-磷酸脱氢酶催化活性,其最适pH值为8,最适反应温度为40℃。2.捻转血矛线虫甘油醛-3-磷酸脱氢酶虫体组织分布的研究为了研究捻转血矛线虫甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, HcGAPDH)在虫体中的分布与定位情况,我们应用常规方法表达已构建好的pET28a-HcGAPDH, GE纯化柱纯化后,免疫SD大鼠制备抗血清。浓缩型S-P免疫组化3步法检测HcGAPDH抗原在捻转血矛线虫体内的分布和表达。结果发现,HcGAPDH主要定位于小肠微绒毛上皮,在其他部位则鲜见。3.捻转血矛线虫pVAX1-HcGAPDH DNA疫苗的构建与体内表达情况的检测利用DNA重组技术,构建pVAX1-HcGAPDH DNA疫苗。经酶切鉴定后,腿部肌肉免疫小鼠,1周后取肌肉注射部位、非注射部位组织,利用RT-PCR和Western-blot技术分别检测DNA疫苗的转录与表达情况。RT-PCR检测结果显示,小鼠注射部位肌肉所扩增出的目的条带大小约为1000bp,非注射部位中未能检测到目的条带,从而说明重组质粒在注射部位可以成功转录；VVestern blot检测结果发现,在注射部位检测到大小为38kD的目的蛋白条带,而非注射部位未能检测到目的蛋白的条带,这些结果都为下一步的动物保护性试验奠定了基础。4.捻转血矛线虫pVAX1-HcGAPDH DNA疫苗的免疫保护性试验使用DNA疫苗pVAX1-HcGAPDH,对9-10月龄山羊做了免疫保护性试验。健康山羊15只,随机分成3组,每组5只。一组免疫100μg pVAX1-HcGAPDH,其他两组为不攻虫对照组和攻虫对照组。在试验山羊的背部皮下多点注射免疫,两个对照组用1m1PBS代替DNA疫苗,一共免疫两次,中间间隔2周。第二次免疫14天后,疫苗免疫组和攻虫对照组山羊口腔接种5000条捻转血矛线虫第三期感染性幼虫。从攻虫后22至34天,每隔一天收集粪便,虫卵计数。攻虫后35天,屠宰山羊并剖解皱胃,分离雌雄虫体,进行成虫计数。同时,采用间接酶联免疫吸附试验(ELISA)方法,测定了所有试验山羊血清特异性IgG滴度、血清以及胃粘膜表面和胃粘膜组织匀浆中特异性IgA浓度。在免疫前、首免后14d、二免后14d、攻虫后14d和杀羊前对以下指标进行检测：利用ELISA试剂盒检测血清细胞因子(IFN-γ、TGF-β、IL-4、IL-22)的浓度变化；利用流式细胞术检测了所有试验山羊外周血中CD4+T淋巴细胞、CD8+T淋巴细胞和B淋巴细胞的变化；利用全自动电子血细胞分析仪对所有试验山羊的外周血中嗜酸性粒细胞、嗜碱性粒细胞、嗜中性粒细胞、单核细胞和血红蛋白进行了检测。结果显示,DNA疫苗pVAX1-HcGAPDH免疫山羊后,ELISA检测结果表明血清IgG在首免后14天达到较高水平,二免后抗体水平进一步升高；血清IgA在免疫后出现较高水平的滴度,同时杀羊后发现胃粘膜组织匀浆具有较高的滴度,免疫后山羊CD4+T淋巴细胞、B淋巴细胞水平显著高于不攻虫对照组,同时细胞因子检测免疫组IL-4水平上升显著,IFN-γ、TGF-β、IL-22变化规律性不明显。分析试验山羊血细胞发现,免疫组和攻虫对照组山羊嗜酸性粒细胞浓度显著高于不攻虫对照组。5000条捻转血矛线虫三期幼虫攻击试验山羊后,免疫组虫卵减少率为34.9%,雌虫、雄虫和成虫总数减少率分别为39.23%,36.03%和37.73%,说明pVAX1-HcGAPDH对抵御山羊捻转血矛线虫的感染具有一定效果。5.捻转血矛线虫烯醇化酶特性的研究根据GenBank上登录的捻转血矛线虫烯醇化酶(Enolase)基因EST序列(登录号为BF422728,登录长度为482bp)设计基因特异性引物,分别利用5’RACE和3’RACE获得该基因5’端未知区和3’端未知区,从而拼接得到Enolase基因的全长cDNA。分析发现：该基因全长1583bp,含有最大开放阅读框(ORF)为1305bp,5’非翻译区(5’-UTR)长62 bp,3’非翻译区(3’-UTR)长21 6bp。根据Enolase基因的全长cDNA设计1对特异性引物,以捻转血矛线虫的总RNA为模板,采用RT-PCR技术,扩增出约1300bp的产物,将该片段克隆到pET-28a(+)载体中,转化入感受态大肠杆菌BL21(DE3)进行表达。SDS-PAGE结果分析发现融合蛋白在上清和包涵体中均有表达,分子量约49kDa。以自然感染的山羊血清作为一抗,Western blot结果显示在49kDa处出现特异性条带。HcGAPDH表达蛋白的包涵体免疫大鼠制备高免血清做一抗,用Western blot分析HcGAPDH基因在捻转血矛线虫的天然表达,结果在49kDa处有明显条带,与推测ORF的蛋白大小近似。酶活性测定结果表明该重组蛋白具有一定的烯醇化酶催化活性,其最适pH值为7。6.捻转血矛线虫磷酸甘油酸激酶的生物信息学分析根据GenBank上登录的捻转血矛线虫磷酸甘油酸激酶(Phosphoglycerate kinase, HcPGK)基因EST序列(登录号为CB192372,登录长度为584bp)设计基因特异性引物,分别利用5’RACE和3’RACE获得该基因5’端未知序列和3’端未知序列,从而拼接得到PGK基因的全长cDNA。利用生物信息学网站的各种在线分析工具,对其进行分析发现：该基因全长1584bp,最大开放阅读框(ORF)为1254 bp,5’非翻译区(5’-UTR)长95 bp,3’非翻译区(3’-UTR)长235 bp。该基因编码417个氨基酸,相对分子量理论预测值为44,594 Da。存在多个潜在的生物活性功能位点,蛋白的理化性质稳定。有17个主要的B细胞抗原表位,这些为下步研究奠定了基础。更多还原

【Abstract】 Haemonchus contortus is a blood-sucking nematode that primarily infects the abomasum of sheep and goats worldwide. Infections with this parasite can cause anaemia, weight loss and death. The current methods for the control of gastrointestinal nematodes rely heavily on the use of chemicals, but this has led to an increase of parasite anthelmintic-resistant and environmental pollution. So we must develop alternative or supplementary means of control, such as molecular vaccines or new anthelmintic chemicals that leave little residue.This research mainly focuses on the characteration of glyceraldehyde-3-phosphate dehydrogenase and its immunoprotection on goats, other research includes the characteration of H. contortus enolase, the immunolocalization of GAPDH in H.contortus, the bioinformatics analysis of H.contortus phosphoglycerate kinase.1. Characterization of adult Haemonchus contortus GAPDH (HcGAPDH)Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expression sequence tag (EST, GenBank accession no.AF670737) to amplify the 3’-and 5’-ends of HcGAPDH. The full length of cDNA from this gene was obtained by overlapping the sequences of 3’-and 5’-extremities and amplification by reverse transcription PCR, then this gene was send to NCBI(accession no.HM145749). The results showed that the cloned full length cDNA comprised 1303bp, a 1026 bp open reading frame (ORF) was found that terminated with the TAA stop codon. The biochemical activities of the recombinant protein HcGAPDH, which was expressed in prokaryotic cells and purified by affinity chromatography, were analyzed by assays of enzymatic activity, thermal stability and pH. The biochemical assay showed that the protein encoded by the HcGAPDH exhibited enzymatic activity with NAD+ as a cofactor. HcGAPDH was stable between pH 5 and 9 and maintained activity at high temperatures of up to 75℃. The natural GAPDH of Haemonchus contortus detected by immunoblot assay was approximately 38 kDa in size, and the recombinant HcGAPDH was recognized strongly by serum from naturally infected goats.2. Histochemistry of the GAPDH in Haemonchus contortus In order to study the localization of the glyceraldehyde-3-phosphate dehydrogenase of Haemonchus contortus (HcGAPDH) in the parasite, the whole paraffin sections of adult worm of H. contortus were studied by immunohistochemical staining with anti-rHcGAPDH serum as the primary antibody. The results showed that GAPDH was expressed in the intestinal tracts of adult female and male worms.3. Construction and identification of DNA Vaccine pVAXl-HcGAPDHFirstly, pVAX1-HcGAPDH DNA vectors were constructed, and then the recombinant DNA plasmids were injected into leg muscle of mice. RT-PCR and Western blot were utilized to analyze the expression of vaccines in mice. The results indicated that DNA vaccines could be well transcripted and expressed in injected tissues.4. Immunoprotective effect of DNA vaccine pVAX1-HcGAPDHFifteen locally bred goats (9-10 months) were raised under nematode-free conditions. The goats were allocated into 3 experimental groups randomly. The H. contortus DNA vaccine was administered 100μg each of pVAX1-HcGAPDH dissolved in lml of PBS (pH7.4). After that, the lml injection volume was equally divided between two injections sites, alternating between the shoulder and thigh muscle. Unchallenged group were not challenged with L3, but vaccinated with lml of PBS of instead, served as the unvaccinated control. Vaccinated animals received two vaccinations at 2 week intervals.5000 infective H. contortus L3 were given two weeks after the final injection. Faecal egg counts were undergone every two days from day 22 after challenge until the end of the experiment. All of the goats were killed 35 days after challenge L3 larvae. Meanwhile, the abomasum was opened immediately, and worm counts were performed. In all goats, serum HcGAPDH specific immunoglobulin IgG titres, IgA titres, mucosal surface (MS) IgA and mucosal homogenate (MH) IgA titres were determined with ELISA. Meanwhile, the peripheral CD4+, CD8+T and B lymphocytes were determined by the method of flow cytometry. The effects of pVAX1-HcGAPDH on cytokine levels of interferon-γ(IFN-γ), IL-4, IL-22 and transforming growth factor-β(TGF-β) of goat peripheral blood were also examined in this study. At last, the peripheral blood eosinophil, basophil, neutrophil, monocyte counts and hemoglobin concentration were determined, respectively. The results showed:After a single L3 larvae challenge of group 1 (pVAX1-HcGAPDH) immunized goats, cumulative mean EPG (eggs per gram of feces) and worm burdens were reduced by 34.9% and 37.73%, respectively. Before L3 challenge infection, the vaccinated groups had significantly higher HcGAPDH-specific IgG titres and IgA titres than the unchallenged and challenged control goats. After L3 challenge, the vaccinated and challenged control groups had significantly higher serum HcGAPDH-specific IgG titres and IgA titre compared to the unchallenged controls. These findings suggested that the HcGAPDH-specific IgG and MH IgA could play a role in protection against H. contortus parasite. Meanwhile, prior to L3 challenge infection, the CD4+ T lymphocytes and B lymphocytes were significantly higher in the vaccinated group as compared to the unchallenged control. After L3 challenge infection, the vaccinated and challenged control groups had higher CD4+ T lymphocytes than the unchallenged control group. Also, we found that the challenged control group had significantly higher B lymphocytes than the vaccinated group. By the determination of cytokine levels, we found the concentration of IL-4 was significantly higher after DNA vaccination. The mean number of blood eosinophils, neutrophil and monocyte increased after immunization coincident with low total worm burdens in the pVAX1-HcGAPDH protected goats.5. Characterization of adult Haemonchus contortus enolase (HcENO)Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expression sequence tag (EST, GenBank accession no.BF422728) to amplify the 3’-and 5’-ends of HcENO. The full length of this gene was obtained by overlapping the sequences of 3’-and 5’-extremities and amplification by RT-PCR. The results, showed that the cloned full length cDNA comprised 1583bp. a 23 bp 5’-untranslated region (5’-UTR) was detected before the ATG initiation codon and a 1305 bp open reading frame (ORF) was found that terminated with the TAA stop codon. On the 3’-end, the cDNA had a 216 bp 3’-UTR with a potential functional polyadenylation signals (AGTAAA) in frame followed by a 13 bp poly-A tail. The biochemical assay showed that the protein encoded exhibited enzymatic activity, the optimal pH for enzymatic activity of recombinant enolases were about pH 7.0. Also, the recombinant HcENO was recognized strongly by serum from naturally infected goats. These results suggested that the enzyme might play potential roles in the infection of H, contortus.6. Bioinformatics analysis of HcPGKThe phosphoglycerate kinase gene of Haemonchus contortus (HcPGK) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expression sequence tag (EST, GenBank accession no.CB 192372) to amplify the 3’-and 5’-ends of HcPGK. After this, some bioinformatics analysis were performed, for example, amino acid and atomic compositions, isoelectric point, extinction coefficient, secondary structure predictions, signal peptide cleavage sites, et al. The results showed that the cloned full length cDNA comprised 1584bp. With bioinformatics analysis, we found that the HcPGK encodes 417 amino acids, molecular weight 44,594 Da, also found that it has a few active domains and motifs.更多还原