动物医学进展
動物醫學進展
동물의학진전
PROGRESS IN VETERINARY MEDICINE
2013年
12期
17-21
,共5页
袁庄川%王鲁彦%梅匀安%张敏%王晓杜
袁莊川%王魯彥%梅勻安%張敏%王曉杜
원장천%왕로언%매균안%장민%왕효두
猪繁殖与呼吸综合征病毒%GP5 基因%原核表达%纯化
豬繁殖與呼吸綜閤徵病毒%GP5 基因%原覈錶達%純化
저번식여호흡종합정병독%GP5 기인%원핵표체%순화
PRRSV%GP5 gene%prokaryotic expression%purification
猪繁殖与呼吸综合征病毒(PRRSV)可引起各种年龄猪的呼吸系统和母猪繁殖机能障碍,导致猪场损失严重,其中 GP5蛋白在 PRRSV 病毒侵入、致病及免疫保护中发挥重要作用。提取猪繁殖与呼吸综合征病毒基因组 RNA,反转录合成 cDNA,PCR 扩增获得 GP5基因,片段大小为600 bp。将 GP5基因亚克隆到原核表达载体 pGEX-6P-1上,菌落 PCR 和限制性内切酶双酶切鉴定,重组质粒 PGEX-6P-1-GP5构建成功,重组质粒转化大肠埃希菌 BL21(DE3),IPTG 诱导表达,SDS-PAGE 分析重组蛋白的表达,Western blot 分析重组蛋白的反应原性。结果表明,重组融合蛋白 His-GP5分子质量约为41 ku,主要以包涵体的形式存在,表达量占菌体总蛋白的35%以上,纯化后的重组蛋白占总蛋白的75%以上;并且该重组蛋白能与猪的阳性血清反应,说明其具有较好的免疫原性。研究结果为基因工程疫苗的研发和 GP5的抗体制备奠定了基础。
豬繁殖與呼吸綜閤徵病毒(PRRSV)可引起各種年齡豬的呼吸繫統和母豬繁殖機能障礙,導緻豬場損失嚴重,其中 GP5蛋白在 PRRSV 病毒侵入、緻病及免疫保護中髮揮重要作用。提取豬繁殖與呼吸綜閤徵病毒基因組 RNA,反轉錄閤成 cDNA,PCR 擴增穫得 GP5基因,片段大小為600 bp。將 GP5基因亞剋隆到原覈錶達載體 pGEX-6P-1上,菌落 PCR 和限製性內切酶雙酶切鑒定,重組質粒 PGEX-6P-1-GP5構建成功,重組質粒轉化大腸埃希菌 BL21(DE3),IPTG 誘導錶達,SDS-PAGE 分析重組蛋白的錶達,Western blot 分析重組蛋白的反應原性。結果錶明,重組融閤蛋白 His-GP5分子質量約為41 ku,主要以包涵體的形式存在,錶達量佔菌體總蛋白的35%以上,純化後的重組蛋白佔總蛋白的75%以上;併且該重組蛋白能與豬的暘性血清反應,說明其具有較好的免疫原性。研究結果為基因工程疫苗的研髮和 GP5的抗體製備奠定瞭基礎。
저번식여호흡종합정병독(PRRSV)가인기각충년령저적호흡계통화모저번식궤능장애,도치저장손실엄중,기중 GP5단백재 PRRSV 병독침입、치병급면역보호중발휘중요작용。제취저번식여호흡종합정병독기인조 RNA,반전록합성 cDNA,PCR 확증획득 GP5기인,편단대소위600 bp。장 GP5기인아극륭도원핵표체재체 pGEX-6P-1상,균락 PCR 화한제성내절매쌍매절감정,중조질립 PGEX-6P-1-GP5구건성공,중조질립전화대장애희균 BL21(DE3),IPTG 유도표체,SDS-PAGE 분석중조단백적표체,Western blot 분석중조단백적반응원성。결과표명,중조융합단백 His-GP5분자질량약위41 ku,주요이포함체적형식존재,표체량점균체총단백적35%이상,순화후적중조단백점총단백적75%이상;병차해중조단백능여저적양성혈청반응,설명기구유교호적면역원성。연구결과위기인공정역묘적연발화 GP5적항체제비전정료기출。
Porcine reproductive and respiratory syndrome virus (PRRSV)can cause obstacle of all age pigs in respiratory system and dysfunction of sow reproduction.The GP5 of PRRSV is important to virus adhe-sion,pathogenicity,immune protection.In this study,we extracted the RNA of PRRSV genome.The full-length GP5 gene was amplified from cDNA of PRRSV by reverse transcription PCR.The GP5 gene fragment is 594 bp.The GP5 gene was subcloned into the prokaryotic expression vector pGEX-6P-1.By colony PCR identification and restriction endonuclease digestion,the recombinant plasmid (pGEX-6P-1-GP5)was transformed into E.coli BL21(DE3),His-GP5 protein was expressed by isopropy-beta-D-thio-galactopyranoside (IPTG )induction,whose molecular weight is 41 ku.The results showed that His-GP5 protein was mainly in the form of inclusion bodies and more than 35% of the total bacterial proteins by SDS-PAGE analysis.The purification products of recombinant protein was more than 75% of the total protein.The recombinant protein has preferably an immunogenic by Western-blot analysis.This research is the foundation to the preparation of the anti-GP5 antibody and genetically engineered vaccine of GP5.