基因组学与应用生物学
基因組學與應用生物學
기인조학여응용생물학
Genomics and Applied Biology
2011年
4期
311-315
,共5页
李运娜%黄金贵%李建华%宫鹏涛%杨举%李赫%李淑红%张西臣
李運娜%黃金貴%李建華%宮鵬濤%楊舉%李赫%李淑紅%張西臣
리운나%황금귀%리건화%궁붕도%양거%리혁%리숙홍%장서신
刚地弓形虫%TgCyP,基因克隆%真核表达
剛地弓形蟲%TgCyP,基因剋隆%真覈錶達
강지궁형충%TgCyP,기인극륭%진핵표체
Toxoplasma gondii%TgCyP%Gene clone%Eukaryotic expression
亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasmngondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用。本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHI和EcoRI酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因。PCR产物连接到pMD18-T克隆载体。用限制性内切酶BamHI和EcoRI双酶切克隆载体,将TgCyP目的基因克隆到载体pVAXI,构建真核表达质粒pVAXl-TgCyP。将真核表达质粒转染Hela细胞,利用问接免疫荧光检测其在Hela细胞内的表达情况。结果表明扩增的TgCyP基因与GenBank上相应基因序列(U04633.1)的一致性达100%,构建的真核表达质粒pVAX1-TgCyP能在转染的Hela细胞中表达,其表达产物与刚地弓形虫阳性血清具有免疫反应性。本研究表明Tg-CyP有望作为弓形虫疫苗的候选抗原,将为进一步研究该质粒的动物免疫试验奠定基础。
親環蛋白(cyclophilin,CyP)是一類廣汎存在于原覈和真覈生物體內的胞溶性蛋白,是剛地弓形蟲(Toxoplasmngondii)速殖子的主要成分,能夠誘導產生IL-12和IFN-γ,在控製弓形蟲急性感染過程中起重要作用。本研究根據GenBank髮錶的TgCyP基因序列,設計併閤成一對包含BamHI和EcoRI酶切位點的引物,以cDNA為模闆,應用PCR技術擴增TgCyP基因。PCR產物連接到pMD18-T剋隆載體。用限製性內切酶BamHI和EcoRI雙酶切剋隆載體,將TgCyP目的基因剋隆到載體pVAXI,構建真覈錶達質粒pVAXl-TgCyP。將真覈錶達質粒轉染Hela細胞,利用問接免疫熒光檢測其在Hela細胞內的錶達情況。結果錶明擴增的TgCyP基因與GenBank上相應基因序列(U04633.1)的一緻性達100%,構建的真覈錶達質粒pVAX1-TgCyP能在轉染的Hela細胞中錶達,其錶達產物與剛地弓形蟲暘性血清具有免疫反應性。本研究錶明Tg-CyP有望作為弓形蟲疫苗的候選抗原,將為進一步研究該質粒的動物免疫試驗奠定基礎。
친배단백(cyclophilin,CyP)시일류엄범존재우원핵화진핵생물체내적포용성단백,시강지궁형충(Toxoplasmngondii)속식자적주요성분,능구유도산생IL-12화IFN-γ,재공제궁형충급성감염과정중기중요작용。본연구근거GenBank발표적TgCyP기인서렬,설계병합성일대포함BamHI화EcoRI매절위점적인물,이cDNA위모판,응용PCR기술확증TgCyP기인。PCR산물련접도pMD18-T극륭재체。용한제성내절매BamHI화EcoRI쌍매절극륭재체,장TgCyP목적기인극륭도재체pVAXI,구건진핵표체질립pVAXl-TgCyP。장진핵표체질립전염Hela세포,이용문접면역형광검측기재Hela세포내적표체정황。결과표명확증적TgCyP기인여GenBank상상응기인서렬(U04633.1)적일치성체100%,구건적진핵표체질립pVAX1-TgCyP능재전염적Hela세포중표체,기표체산물여강지궁형충양성혈청구유면역반응성。본연구표명Tg-CyP유망작위궁형충역묘적후선항원,장위진일보연구해질립적동물면역시험전정기출。
Cyclophilins (CyPs) are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-12 and IFN-γ and may be play an important part in the process of controlling acute phase oftoxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH I and EcoR I restriction enzyme site. The eDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector. After cloned, the vector was digested with BamH I and EcoR I and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAX1-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.