【作者】 周前进；

【导师】 杜爱芳；

【作者基本信息】 浙江大学 ， 预防兽医学， 2010， 博士

【摘要】 捻转血矛线虫(Haemonchus contortus)是牛、羊等反刍动物的主要寄生性线虫,寄生于动物皱胃粘膜的表面,引起捻转血矛线虫病,导致动物出现贫血、消瘦,甚至引起死亡。使用抗蠕虫药物是目前防治该病的主要措施,但线虫耐药性的日益严重迫切需要发展新的防治策略,疫苗作为一种新的有效的防治策略,具有广阔的应用前景。由于线虫具有复杂的生活史,抗原成分繁多,到现在为止,还没有一种线虫疫苗应用于生产。氨肽酶H11是捻转血矛线虫L4幼虫和成虫阶段表达的一种整合膜蛋白,被认为是目前为止免疫效果最好的一种候选抗原(减卵率>90%,减虫率>75%),而利用大肠杆菌和昆虫杆状病毒系统表达的重组形式均不能引起有效的免疫保护。秀丽隐杆线虫因其与寄生性线虫在进化上的近缘性,被认为是研究寄生性线虫基因功能和表达寄生性线虫疫苗候选抗原的理想系统。本研究的主要目的是：探明捻转血矛线虫疫苗候选抗原基因H11的基因组结构特征；同时以秀丽隐杆线虫为模式,建立研究寄生性线虫基因功能和表达寄生性线虫疫苗候选抗原的转基因技术体系；在此基础上分析H11基因5’侧翼区序列的启动子转录活性,研究利用建立的转基因技术体系,表达H11基因的可行性,为寄生性线虫的疫苗研制提供新方法。1.捻转血矛线虫疫苗候选抗原基因H11的基因组结构分析参照已发表的捻转血矛线虫疫苗候选抗原H11的基因序列,以捻转血矛线虫ZJ株的总RNA为模板,进行RT-PCR扩增,成功扩增出H11基因。对H11及其同源类似物进行比对分析,发现H11含有一个锌指结合基序HEXXHXW,具有典型的微粒体氨肽酶结构域特征。通过在功能域保守区域设计特异性引物,利用长距离PCR技术和基因步移技术,成功获得了H11的基因组序列。结果表明,H11的ORF大小为2919 bp,编码972个氨基酸；获得的H11基因组大小为14959 bp,拥有25个外显子和24个内含子,内含子和外显子之间具有典型的GT……AG结构。捻转血矛线虫H11基因组序列的获得为我们进一步开展H11的相关研究提供了重要素材。2.以秀丽隐杆线虫为模式,建立研究寄生性线虫基因功能和表达寄生性线虫疫苗候选抗原的转基因技术体系为建立秀丽隐杆线虫的转基因技术体系,利用PCR技术从秀丽隐杆线虫基因组DNA中扩增获得Act-1基因的核心启动子序列、T07F10.lA基因的核心启动子序列与3’UTR的Poly (A)信号序列,以及从表达载体pEGFP-N1上扩增获得SV40早期mRNA的Poly (A)信号序列与EGFP基因。以pBluescript SK+或pEGFP-4.1为基本载体骨架,分别构建由Act-1基因的核心启动子序列、T07F1O.lA基因的核心启动子序列调控,EGFP作为报告基因的重组表达载体。通过显微注射将重组载体与Marker质粒pRF4共注射到秀丽隐杆线虫性腺,结果发现重组载体Pact-EGFP中Act-1基因的核心启动子能够指导EGFP在秀丽隐杆线虫的皮层、副皮层以及咽部表达。利用脂质体介导转染Vero细胞,发现重组载体Pact-EGFP和pB-Pact-EGFP-SV40T中的核心启动子能够指导EGFP在Vero细胞中表达,但表达强度具有较大的差异,表明秀丽隐杆线虫Act-1基因的核心启动子区域上游以及基因下游的转录终止信号区域可能存在与转录水平密切相关的独特的转录调节元件。该研究为进一步实现寄生性线虫基因在秀丽隐杆线虫体内的表达奠定了基础。3.利用建立的转基因技术体系,分析H11基因5’侧翼区序列的启动子转录活性为进一步分析H11基因的5’侧翼区的转录活性,在获得的H11基因组序列的基础上,再次利用基因步移技术扩增5’上游的部分基因组序列,确认H11基因5’侧翼区大小为1517 bp；同时获得了H11亚型H11-4最后一个外显子和3’UTR序列,发现H11-4基因与H11基因在染色体上不仅是连锁的,而且具有相同的基因延伸方向。将获得的5’侧翼区序列单独或联合H11基因的前两个外显子、第一内含子以及第二内含子的部分序列克隆到表达载体ppd95.77 GFP基因的上游,显微注射到线虫的性腺。结果表明H11基因5’侧翼区序列能够指导GFP在秀丽隐杆线虫L4幼虫和成虫肠管的前端和末端特异性表达,且显示出与H11在捻转血矛线虫体内不完全相同的表达模式；H11的部分外显子和内含子序列在秀丽隐杆线虫体内不能实现正确的剪接,这可能与H11是捻转血矛线虫寄生阶段特异性表达的蛋白有关。4.利用建立的转基因技术体系,筛选用于指导H11疫苗候选抗原表达的启动子序列,并在该启动子的指导下实现H11基因在秀丽隐杆线虫体内的表达选择秀丽隐杆线虫体内H11的同系物T07F1O.1a基因以及肠道特异性表达的cpr-1和elt-2基因的5’侧翼区序列作为筛选对象,用以在秀丽隐杆线虫体内表达H11疫苗候选抗原。T07F10.1α、cpr-1和elt-2基因的1885 bp、1985 bp和1998 bp的5’侧翼区序列克隆到表达载体ppd95.77 GFP基因的上游,显微注射到线虫的性腺。结果显示,在T07F10.1a基因的5’侧翼区的转录下, GFP能够在秀丽隐杆线虫除去胚胎期的整个生命周期中都有表达,表达部位主要集中在秀丽隐杆线虫的尾部神经元、咽部、分泌细胞、肠道细胞以及神经系统；在cpr-1基因的5’侧翼区的转录下,GFP能够特异性的在线虫的整个肠道表达,尤其在L4幼虫和成虫阶段,GFP呈现较高强度的表达；在elt-2基因的5’侧翼区的转录下GFP能够在L3幼虫到成虫阶段的肠道表达,且呈现明显的区段性,即肠道最前端的肠环1(int1,前肠)、中肠(肠道最中间的部分)以及肠道末端,尤其int1位置的GFP呈现较高强度的表达,但表达强度与部位因虫体个体不同有较大差异。根据GFP表达的组织定位、L4幼虫和成虫阶段GFP的表达强度以及虫体表达GFP的稳定性等差异,最后采用cpr-1的5’侧翼区序列作为在秀丽隐杆线虫表达H11的启动子。在已获得的重组载体cpr-1 5’侧翼区∷GFP基础上,利用特异性引物ppdF/ppdR,在高保真酶作用下,通过长距离PCR扩增切除GFP基因,进行载体自连、环化,同时在载体上引入SacⅠ.SaccⅡ等4个酶切位点构建重组表达载体cpr-pPD95.77(G-)；通过引入EGFP基因对重组载体验证后,利用SacⅠ和SacⅡ限制性内切酶将基因两端加有6×His标签的H11及其部分基因片段Trans-HSP分别引入重组表达载体。cpr-pPD95.77(G-)中crp-1 5’则翼区能够指导EGFP在秀丽隐杆线虫的肠道特异性表达表明改造后的重组载体在秀丽隐杆线虫体内具有表达外源基因的功能。Western-blot分析结果显示,在cpr-1 5’侧翼区的启动下,H11基因在秀丽隐杆线虫体内没有明显表达,而Trans-HPS能够在秀丽隐杆线虫体内表达；尽管His单克隆抗体与多克隆抗体能够检测到大小一致的特异性目的条带,利用镍琼脂糖凝胶的方法却未能实现对Trans-HPS的纯化,因此,我们选择Trans-HPS转基因线虫的全虫抗原作为免疫原进行动物免疫保护性试验。5.利用获得的转基因蛋白免疫山羊,比较分析免疫保护效果采用液体培养的方法大量获取转基因表达Trans-HPS蛋白的秀丽隐杆线虫成虫,制备全虫抗原,以250μg/只的剂量免疫山羊,加强免疫一次,用捻转血矛线虫L3幼虫5000条/只的感染强度感染山羊,结合各种指标检测含有Trans-HPS蛋白的全虫抗原的免疫保护效果。ELISA检测结果表明首免后两周既可检测到较高的IgG抗体水平,二免后两周达到高峰,二免后7周抗体水平仍可维持较高水平；外周血淋巴增殖试验结果显示,Trans-HSP蛋白能够显著增强Trans-HPS蛋白免疫组(p<0.01)和HPS蛋白免疫组(p<0.05)淋巴细胞的增殖活性；对不同免疫组山羊排出虫卵数、幼虫孵化率和成虫数进行统计分析,发现Trans-HPS蛋白免疫组减卵率为41.4%,且能明显延缓排卵高峰期的到来；Trans-HPS蛋白免疫组成虫减少率为17.79%,与感染阳性组比较差异不显著。综上,本研究首次获得了H11的基因组序列,发现了亚型H11-4与H11之间的基因连锁现象；建立了以秀丽隐杆线虫为模式的转基因技术体系,利用该体系成功实现了H11部分基因片段Trans-HPS的转基因表达,并对该转基因表达蛋白的免疫保护性进行了评估。本研究为开发寄生性线虫疫苗提供了新技术,为进一步研究寄生性线虫的基因功能奠定了理论基础。更多还原

【Abstract】 The gastrointestinal nematode Haemonchus contortus is a major blood feeding pathogen of sheep and goats, which inhabits the mucosal surface of the abomasum in these ruminants and causes haemonchosis. Its infections may be manifested as anemia, weight loss or even death in some cases. Current control of Haemonchosis mainly depends on the use of anthelmintic drugs. But the increasing problem of resistance to all classes of anthelmintics highlights the need for novel control strategy, such as vaccination. No vaceine has yet been commercialized, due to the complicated life-cycle and incomprehensible components of antigens. Aminopeptidase H11 is an integral membrane glycoprotein which is present only in the intestinal microvilli of L4 and adults worms, and considered to be one of the best characterized hidden antigens from H. contortus (on average a greater than 90% reduction in faecal egg counts and a greater than 75% reduction in worm burdens). But no effective protection was provided by vaccination with the recombinant forms expressed in E. coli or baculovirus. The free-living nematode Caenorhabditis elegans has been validated as an alternative expression system to attempt to express proteins similar to their native forms, as it is closely related to H. contortus in evolutionary terms. Here, the study was aimed to characterizing genomic structure of Hll gene from H. contoruts, and to establish a transgenic platform for functional exploitation of genes and expression of antigen candidates from parasitic nematodes, taking C. elegans as a heterologous system, then to estimate the transcriptional activity of H11 5’ flanking region and make sure the feasibility in expression of Hll using C. elegans, which could provide a novel strategy for vaccination against parasitic namatodes.1. Characterization of genomic structure of Hll gene from H. contorutsReferred to the sequence of Hll from H. contortus reported by Smith (1997), RT-PCR was carried out to amplify the cDNA of Hll, taking total RNA of H. contortus as a template. Alignment with its similarities from other organisms revealed a typical characterization of microsomal aminopeptidase with a zinc-binding motif HEXXHXW. Specific primers were designed, according to the conserved domains among these similarities, to amplify the genomic DNA of H11, employing the long-distance PCR and genome walking technique. The results revealed the open reading frame of H11 was 2919 bp in size, while the genomic DNA was 14,959 bp in size which contained 25 exons separated by 24 introns, and canonical GT-AG consensus sequences were at each intron-exon splice junction. These findings provided interesting materials to our further research.2. Establishment of a transgenic platform to exploitation of gene function and vaccine development of parasitic nematodes, taking C. elegans as a heterologous systemTo establish the transgenic platform, the core promoter regions of Act-1 gene and T07F10 gene, and the Poly (A) signal region of T07F10 gene were amplified from the genomic DNA of C. elegans using PCR, and SV40 early mRNA poly(A) signal sequence and EGFP gene were amplified from eukaryotic vector pEGFP-N1, respectively. Taking the vector pBluescript SK+ or pEGFP-4.1 as backbone, several varied recombinants were constructed, three of which, that is recombinant Pact-EGFP, pB-Pact-EGFP-T07F10T and pB-Pact-EGFP-SV40T, contained the core promoter region of Act-1 gene, and the others, named as recombinant pB-PT07F10-EGFP-T07F10T and pB-PT07F10-EGFP-SV40T, contained the core promoter region of T07F10 gene. After microinjection into the gonad of C. elegans together with pRF4 as a gene marker, only the core promoter region of Act-1 gene in vector Pact-EGFP could direct the expression of EGFP and green fluorescence was detected in the cortex, vice cortex and the pharyngeal of C. elegans. In contrast, the core promoter region of Pact-EGFP and pB-Pact-EGFP-SV40T could direct the expression of EGFP in Vero cell with different intensity, when transfected into Vero cell by liposome, which indicated that unique regulatory elements to transcription may exist in the 5’upstream of the core promoter or the 3’UTR of the gene. These results prompted greatly the further research on gene function of parasitic nematodes using C. elegans.3. Characterization of transcriptional activity of Hll 5’-flanking region, taking advantage of C. elegansTo proceed characterizing the transcriptional activity of Hll 5’-flanking region, genome walking was employed again to obtain more information of Hll 5’upstream. Alignment revealed the 1517-bp 5’-flanking region of Hll, and the last exon and its 3’ UTR of an isoform H11-4. The result indicated that H11-4 had a tandem link to Hll gene, with the same orientation in gene extension in the chromosome. The 1517-bp 5’-flanking region of Hll was cloned upstream of GFP gene in ppd95.77 vector only or fused with the first two exons, the first intron and part of the second intron. After microinjected recombinant plasmid with 5’-flanking region of H11 into the gonads of C. elegans, the transformed animals exhibited fluorescence in the most anterior intestine and distal intestine in the L4 larvae and adult worms, which demonstrated different transcriptional patterns compared with that in H. contortus. The correct splice did not occur in C. elegans, when the first two exons, the first intron and part of the second intron of Hll gene was clone behind 5’-flanking region of H11, which was probably correlated with its unique function to the parasitism of H. contortus.4. Screening of promoters used to direct expression of heterologous gene and transgenic expression of Hll gene regulated by cpr-1 5’-flanking region in C. elegansThe 5’-flanking regions of three genes from C. elegans, that is T07F10.1a gene which codes the similarity of H11, cpr-1 and elt-2 gene which are only expressed in intestine, were screened to direct the expression of H11 in C. elegans. The 1885-bp, 1985-bp,1998 bp 5’-flanking region of these three genes were amplified and cloned upstream of GFP gene in pPD95.77 vector. After microinjection into the gonad of C. elegans, the progenies of trangenic worms were picked out and investigated. The results showed that the fluorescence directed by T07F10.1a 5’-flanking region is distinctively expressed in the intestine, excretory cells, nervous system and tail neurons in C. elegans of all stages but embryonic development, while directed by cpr-1 and elt-2 5’-flanking region were only detected in intestine, particularly in the L4 and adult C. elegans. cpr-1 5’-flanking region can direct intensive expression of GFP in the whole intestine. elt-2 5’ flanking region direct the expression of GFP in the most-anterior intestine (int 1), mid-intestine and the distal intestine, and various differences in fluorescence intensity were shown among different individuals. cpr-1 5’-flanking region was determined to direct the expression of H11 gene, referred to tissue location of GFP, expression intensity in L4 and adult C. elegans and stability among different individuals.To obtain expression vector cpr-pPD95.77(G-), a specific pair of primers were designed. Taking advantage of PrimeStarTM SH DNA polymerase, GFP gene in recombinant vector cpr-1 5’-flanking region::GFP was removed employing long-distance PCR. The vector cpr-pPD95.77(G-) was formulated after self-ligation and cyclization of the amplification product with addition of four restriction enzymes. EGFP gene was subcloned downstream of cpr-1 5’-flanking region to verify the validation in expression of heterologous genes. Then, H11 gene and its partial sequence were cloned downstream of cpr-1 5’-flanking region into cpr-pPD95.77(G-), respectively, with 6 fold His tag added at both sides of the genes. SDS-PAGE and Western-blot were introduced to analyze the expression of both genes. Green fluorescence could be detected in the intestine at a high level, which indicated that recombinant vector cpr-pPD95.77(G-) also had the ability to express heterologous genes. The results suggested that it was not H11 but Trans-HPS could expressed in C. elegans, at the direction of cpr-1 5’-flanking region and specific bands termed Trans-HPS could be detected using both anti-His monoclonal antibody and polyclonal antibody, although Trans-HPS could not be purified using Nickel agarose gel affinity chromatography.5. Assessment of the immuno-protection of Trans-HPS against H. contortus in goatsLiquid culture was employed to bulk preparation of crude extraction from transgenic worms with Trans-HPS.250μg per goat of crude extraction was administered at primary immunization and booster two weeks later with the same dose. Assessment of the immuno-protection proceeded after orally challenged with 5000 infective H. contortus L3 2 weeks after the second immunization. High titre level of serum IgG was mounted just two-week post-primary immunization. Serum IgG levels reached peak values at two-week post-second immunization and remained significant titre until the end of this trial. Significant elevation in T cell proliferative responses stimulated by Trans-HPS was observed in both Trans-HPS immunized group and HPS immunized group. Faecal egg counts was reduced by 41.4% and the advent of the peak in FEC was markedly delayed in Trans-HPS immunized group. And worm burdens were reduced by 17.79%, which showed no statistical significance as positive control.In summary, the genomic DNA of H11 was first sequenced and characterized, and the tandem relationship between Hll gene and its isoform H11-4 was first revealed in our study; transgenic platform for exploitation of gene function and vaccine development of parasitic nematodes was developed and expression characterization and immuno-protection assessment of Trans-H11 proceeded, taking advantage of C. elegans. This system will be particularly important for expression of complex glycan epitopes and examination of their influence on the immune response to parasitic nematodes.更多还原