摘要
为了获得华支睾吸虫成虫期强反应原性抗原基因，首先利用λZAP载体构建华支睾吸虫成虫cDNA表达文库：即从我国东北疫区（镇赉县）家犬胆管内分离收集华支睾吸虫成虫，采用Trizol Reagent提取其总RNA，Oligo（dT）纤维素柱纯化mRNA，反转录合成第1链eDNA及第2链cDNA，用CHROMASPIN-400柱离心层析纯化后，与载体xZAPExpress连接，体外包装后成功获得我国东北疫区华支睾吸虫成虫cDNA表达文库。文库容量为1．5×10^6pfu，重组率为99％，插入片段长度在0．4～2．0kb之间，扩增文库的滴度为1．5×10^10 pfu／mL。然后利用免疫学方法对该cDNA表达文库进行筛选：以自然感染华支睾吸虫的人血清为抗体探针，从2．0×10^5个重组噬菌体筛选强反应原性抗原基因，对筛选出的强反应原性克隆进行测序，利用相关分子生物学软件进行序列分析。共获得41个阳性克隆，测序结果分析表明，这些cDNAs根据其编码的蛋白可分为以下几种，即与来自华支睾吸虫的甘氨酸-2、脯氨酸-2及Cs44抗原高同源的基因及分别与来自Nematostella vectensis的未知蛋白、转录延伸因子及果蝇CG3446基因编码蛋白较低同源性的基因。本研究结果为进一步对华支睾吸虫抗原的生物学特性研究及应用奠定了理论基础和实验依据。
In order to screen antigenic genes of Clonorchis sinensis （C. sinensis）. Firstly, a cDNA expression library from adult worms of C. sinensis was successfully constructed with lambda ZAP express vector. Total RNA of C. sinensis adult worms were extracted by the Trizol reagent and mRNA were further purified through oligo-dT cellulose. The first strand cDNA was synthesized by using MMLV reverse transcriptase. After the synthesis of the second strand,the cDNA were purified by CHROMA SPIN-400 kit and then ligated with lambda ZAP express vector, then packaged in vitro and amplified. The original library contained 1.5 ×10^6 pfu cDNA clones, the titer of amplified library reached 1.5×10^10 pfu/mL,in which about 99 % clones were recombinants and most of insert DNA fragments were 0. 4-2.0 kb. Secondly, immunoscreened using the naturely infected man serum from C. sinensis. The positive clones were sequenced and analyzed. From 2.0× 10^5 recombinant clones of the cDNA library,41 positive clones were obtained. Sequence analysis indicated that the cDNAs encoded proteins including glycine rich antigen 2, proline rich antigen 2 and antigen Cs44 from C. sinensis,anothers were lower similarity to predicted protein from Nematostella vectensis,transcription elongation factor GreA from Bartonella quintana str. Toulouse and NM_ 132090 CG3446 gene product in Drosophila melanogaster from Schistosoma japonicum. These data may form a foundation for identifying recombinant antigens that can be used in the diagnosis or vaccination against clonorchiasis.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2009年第7期868-872,共5页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(NSFC30700600)
吉林省科技厅基金资助项目(20060417)
关键词
华支睾吸虫
成虫
CDNA文库
Clonorchis sinensis
adult larvae
cDNA library
作者简介
吴秀萍（1975-），女，讲师，博士。
通讯作者
通讯作者
