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利用酵母双杂交解析WSSV感染凡纳滨对虾途径的研究
谢世筠
Subtype博士
Thesis Advisor相建海
2016-05-21
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline海洋生物学
Abstract作为最重要的海洋水产养殖种类之一,凡纳滨对虾(Litopenaeus vannamei)的养殖产量占据了世界甲壳动物养殖产量的半壁江山(2013年,49.4%)。但是自上世纪九十年代起,伴随着对虾养殖业的蓬勃发展,各类病害如白斑综合症病毒(WSSV)等威胁也与日俱增。现有研究表明,消化道和鳃是WSSV侵入对虾体内的重要关口,其上可能存在与WSSV相互作用的蛋白。为了了解WSSV的侵染机制,最终找到防治WSSV办法,在本研究中,我们利用酵母双杂交系统寻找与WSSV囊膜蛋白存在相互作用的凡纳滨对虾消化道和鳃组织蛋白,研究内容和结果如下:
1、构建了凡纳滨对虾消化道酵母双杂交文库。提取凡纳滨对虾消化道总RNA用于构建消化道cDNA初级文库,以此初级文库为cDNA来源构建了凡纳滨对虾消化道酵母双杂交文库。将文库菌液稀释后涂布在SD/-Leu平板上,通过计数推算出文库总克隆数约为 。从SD/-Leu平板上随机挑取20个克隆进行平均插入片段长度鉴定,结果显示插入片段平均长度大于1.2 kb。文库总克隆数与插入片段平均长度结果显示构建的凡纳滨对虾消化道酵母双杂交文库符合后续筛选要求,达到了预期目标。
2、构建了凡纳滨对虾鳃酵母双杂交文库。提取凡纳滨对虾鳃总RNA用于构建鳃双链cDNA文库,并对构建的双链cDNA文库进行了均一化处理。通过二轮均一化处理,成功的将高丰度基因含量降低约32倍。随后利用均一化后的文库为cDNA来源构建了凡纳滨对虾鳃酵母双杂交文库。将文库菌液稀释后涂布在SD/-Leu平板上,通过计数推算出文库总克隆数约为 。从SD/-Leu平板上随机挑取24个克隆进行平均插入片段长度鉴定,结果显示插入片段平均长度大于800 bp。文库总克隆数与插入片段平均大小结果显示构建的凡纳滨对虾鳃酵母双杂交文库符合后续筛选要求,达到了预期目标。
3、尝试构建WSSV囊膜蛋白VP13B、VP19、VP24、VP26、VP28、VP31、VP32、VP37、VP38A、VP39B 、VP41A、VP41B、VP51A、VP51B、VP60A和VP73的诱饵菌株,发现VP13B、VP26和VP31存在自激活作用,其余13个WSSV囊膜蛋白的诱饵菌株均可用于酵母双杂交筛选。随后以VP28、VP37和VP51A作为诱饵蛋白,分别与凡纳滨对虾消化道酵母双杂交文库和鳃酵母双杂交文库进行酵母双杂交筛选。通过筛选、序列比对和与课题组的凡纳滨对虾转录组数据比对分析,从凡纳滨对虾消化道酵母双杂交文库获得了60个阳性克隆,插入片段可以比对上24条不同的Unigene,从凡纳滨对虾鳃酵母双杂交文库获得了51个阳性克隆,插入片段可以比对上22条不同的Unigene。经过分析,在筛选结果中发现1条Unigene可注释为peritrophin [Litopenaeus vannamei],命名为LvPT。LvPT含有276个氨基酸,1-19位氨基酸为信号肽,在90-149、151-198和200-257位氨基酸各有1个的2型几丁质结合结构域。
4、在酵母双杂交系统中重新验证了LvPT与VP37的相互作用,并发现LvPT还分别与VP32、VP38A、VP39B和VP41A在酵母双杂交系统中存在相互作用;通过免疫共沉淀实验进一步确认了LvPT与VP37在酵母双杂交系统中的结果。根据LvPT的结构域功能实验和半定量PCR结果推测,LvPT在凡纳滨对虾体内极有可能是一种具有外分泌特性的几丁质结合蛋白,并且参与了凡纳滨对虾围食膜的形成,是凡纳滨对虾围食膜重要的结构蛋白之一。
5、合成LvPT的肽段(CKQEGRYPDLLNQQ)作为抗原免疫新西兰大白兔,获得了LvPT的多克隆抗体。抗体检测结果显示在凡纳滨对虾胃总蛋白中可以特异性地检测到LvPT,条带大小在27 kDa左右。随后使用双链RNA干扰技术在转录水平沉默LvPT表达后,采用反向灌肠作为攻毒方式进行WSSV攻毒实验。攻毒实验结果显示,在攻毒48h和72h后,实验组对虾(LvPT dsRNAi+WSSV)游泳足、胃和肠组织中的病毒拷贝数显著低于对照组(EGFP dsRNAi+WSSV),说明在LvPT表达受到抑制后,WSSV在消化道内侵染受到了明显的抑制,提示在WSSV侵染对虾消化道的过程中,LvPT可能作为吸附因子,辅助病毒粒子完成侵染。
Other AbstractThe quantity of aquaculture production of Pacific white shrimp Litopenaeus vannamei nearly takes half of quantity of aquaculture production of all crustacean species in world (2013, 49.4%). With the development of shrimp aquaculture, pathogens, especially white spot syndrome virus (WSSV), are becoming more and more dangerous. Previous researches indicated that the digestive tract and gill were the portal of WSSV infection. Therefore, the cell membrane of the digestive tract and gill may have some important factors influencing the WSSV infection. In order to understand the mechanism of WSSV infection and solve the threat of WSSV finally, we used yeast two hybrid (Y2H) to investigate the interactions between envelop proteins of WSSV and shrimp proteins in present sudies.
The main progresses are as follows in this dissertation:
1. The Y2H library of L. vannamei digestive tract (library-dt) was constructed. Total RNA was extracted from digestive tract and used for construction of cDNA library. The cDNA library was used for construction of library-dt later. After library construction, the CFU of the library-dt was examined by spreading the diluted library-dt on SD/-Leu plate. The CFU of library was around . Twenty clones were then randomly picked from the SD/-Leu plate and used for average insert fragment length examination. The average insert fragment length of library-dt was longer than 1.2 kb. Both CFU and average insert fragment length of library-dt fulfill the requirement of Y2H screening.
2. The Y2H library of L. vannamei gill (library-g) was constructed. Total RNA was extracted from gill and used for construction of double stand cDNA library. The cDNA library was normalized before it was used for construction of library-g later. The content of high expression level genes was reduced about 32 times. Then, the CFU of the library-g was examined by spreading the diluted library-g on SD/-Leu plate. The CFU of library was around . Twenty four clones were then randomly picked from the SD/-Leu plate and used for average insert fragment length examination. The average insert fragment length of library-g was longer than 800 bps. Both CFU and average insert fragment length of library-g fulfill the requirement of Y2H screening.
3. Thirteen bait strains of envelop proteins of WSSV including VP19, VP24, VP28, VP32, VP37, VP38A. VP39B. VP41A. VP41B. VP51A. VP51B. VP60A and VP73were constructed. VP28, VP37 and VP51A were used as baits in Y2H screening of library-dt and library-g. After screening, 60 positive clones belonging to 24 unigenes were obtained from library-dt; 51 positive clones belonging to 22 unigenes were obtained from library-g. A ueigene named LvPT was obtained from results of library-dt after bioinformatics analysis. The full length of deduced amino acid sequence of LvPT was 276 and LvPT had high identity with peritrophin [Litopenaeus vannamei]. There was a signal peptide located at aa 1-19 and 3 ChtBD2 at aa 90-149, 151-198 and 200-257.
4. The results of the tissue distribution analysis revealed that LvPT was mainly expressed in stomach. The interaction between VP37 and LvPT was confirmed in Y2H system and further study showed that LvPT could also have interactions with VP32, VP38A, VP39B and VP41A in Y2H system. CO-IP assay confirmed the interaction between VP37 and LvPT. The results of domain function assays showed that LvPT was a secretory protein with high chitin binding activity. These results indicated that LvPT might be a structure protein of peritrophic membrane and take part in its formation.
5. Synthetic peptide (CKQEGRYPDLLNQQ) was used as antigen in LvPT antibody production. A band around 27 kDa from total protein of L. vannamei stomach was identified in binding specificity test. WSSV challenge experiment was performed after the dsRNA interference of the expression of LvPT in both transcription and protein level and WSSV solution was injected by reverse gavage. According to the results of qPCR, at 48 and 72 hpi, the WSSV amounts of LvPT dsRNAi + WSSV group were significant lower than EGFP dsRNAi + WSSV group in pleopod, stomach and gut, respectively. The results indicated that the reduction of LvPT could lead to less WSSV infection in digestive tract and LvPT might play a role as attaching factor during WSSV infection in digestive tract.
Subject Area海洋生物学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/112540
Collection实验海洋生物学重点实验室
Affiliation中国科学院海洋研究所
Recommended Citation
GB/T 7714
谢世筠. 利用酵母双杂交解析WSSV感染凡纳滨对虾途径的研究[D]. 北京. 中国科学院大学,2016.
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