CAJ | 학술논문

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栽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cDNA及第2链cDNA,用CHROMA SPIN-400柱离心层析纯化后,与栽体λZAP Express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1.5×106pfu,重组率为99%,插入片段长度在0.4～2.0 kb之阀,扩增文库的滴度为1.5×1010pfu/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2.0×105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.
위료획득화지고흡충성충기강반응원성항원기인,수선이용λZAP재체구건화지고흡충성충cDNA표체문고:즉종아국동북역구(진뢰현)가견담관내분리수집화지고흡충성충,채용Trizol Reagent제취기총RNA,Oligo(dT)섬유소주순화mRNA,반전록합성제1련cDNA급제2련cDNA,용CHROMA SPIN-400주리심층석순화후,여재체λZAP Express련접,체외포장후성공획득아국동북역구화지고흡충성충cDNA표체문고.문고용량위1.5×106pfu,중조솔위99%,삽입편단장도재0.4～2.0 kb지벌,확증문고적적도위1.5×1010pfu/mL.연후이용면역학방법대해cDNA표체문고진행사선:이자연감염화지고흡충적인혈청위항체탐침,종2.0×105개중조서균체사선강반응원성항원기인,대사선출적강반응원성극륭진행측서,이용상관분자생물학연건진행서렬분석.공획득41개양성극륭,측서결과분석표명,저사cDNAs근거기편마적단백가분위이하궤충,즉여래자화지고흡충적감안산-2、포안산-2급Cs44항원고동원적기인급분별여래자Nematostella vectensis적미지단백、전록연신인자급과승CG3446기인편마단백교저동원성적기인.본연구결과위진일보대화지고흡충항원적생물학특성연구급응용전정료이론기출화실험의거.
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 eDNA 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 × 106 pfu cDNA clones,the titer of amplified library reached 1.5 × 1010 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 × 105 recombinant clones of the eDNA 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.