农业科学与技术（英文版）
農業科學與技術（英文版）
농업과학여기술（영문판）
AGRICULTURAL SCIENCE & TECHNOLOGY
2010年
5期
96-100
,共5页
牛瑟氏泰勒虫%重组P23表面蛋白%大肠杆菌%表达条件
牛瑟氏泰勒蟲%重組P23錶麵蛋白%大腸桿菌%錶達條件
우슬씨태륵충%중조P23표면단백%대장간균%표체조건
Theileria sergenti%P23 major surface protein gene%Prokaryotic expression
[目的]研究牛瑟氏泰勒虫P23表面蛋白基因的克隆及原核表达.[方法]采用PCR方法扩增牛瑟氏泰勒虫中国延边株P23基因片段,将扩增产物克隆人pMD18-T载体构建重组质粒pMD18-P23经PCR、双酶切鉴定后测序;将目的基因片段亚克隆人表达载体pGEX-4T-1构建重组表达质粒pGEX-4T-P23,转化宿主菌BL21获得重组菌.通过对诱导条件的优化,根据SDS-PAGE确定表达蛋白的最佳表达条件;Western-blotting检测表达蛋白的反应原性.[结果]所克隆的牛瑟氏泰勒虫P23基因片段长507 bp,与牛瑟氏泰勒虫日本株P23基因的核苷酸同源性达99.4%,表达的融合蛋白大小约为46 ku;诱导时机以接种培养后2 h为最佳,诱导时间以6 h为最佳,诱导温度以34℃为最佳,0.008～1.000 mmol/L的IPTC对表达量的影响不大.Western blotting检测表明该蛋白具有较好的抗原性.[结论]为牛瑟氏泰勒虫病的免疫学诊断和预防等研究奠定了基础.
[目的]研究牛瑟氏泰勒蟲P23錶麵蛋白基因的剋隆及原覈錶達.[方法]採用PCR方法擴增牛瑟氏泰勒蟲中國延邊株P23基因片段,將擴增產物剋隆人pMD18-T載體構建重組質粒pMD18-P23經PCR、雙酶切鑒定後測序;將目的基因片段亞剋隆人錶達載體pGEX-4T-1構建重組錶達質粒pGEX-4T-P23,轉化宿主菌BL21穫得重組菌.通過對誘導條件的優化,根據SDS-PAGE確定錶達蛋白的最佳錶達條件;Western-blotting檢測錶達蛋白的反應原性.[結果]所剋隆的牛瑟氏泰勒蟲P23基因片段長507 bp,與牛瑟氏泰勒蟲日本株P23基因的覈苷痠同源性達99.4%,錶達的融閤蛋白大小約為46 ku;誘導時機以接種培養後2 h為最佳,誘導時間以6 h為最佳,誘導溫度以34℃為最佳,0.008～1.000 mmol/L的IPTC對錶達量的影響不大.Western blotting檢測錶明該蛋白具有較好的抗原性.[結論]為牛瑟氏泰勒蟲病的免疫學診斷和預防等研究奠定瞭基礎.
[목적]연구우슬씨태륵충P23표면단백기인적극륭급원핵표체.[방법]채용PCR방법확증우슬씨태륵충중국연변주P23기인편단,장확증산물극륭인pMD18-T재체구건중조질립pMD18-P23경PCR、쌍매절감정후측서;장목적기인편단아극륭인표체재체pGEX-4T-1구건중조표체질립pGEX-4T-P23,전화숙주균BL21획득중조균.통과대유도조건적우화,근거SDS-PAGE학정표체단백적최가표체조건;Western-blotting검측표체단백적반응원성.[결과]소극륭적우슬씨태륵충P23기인편단장507 bp,여우슬씨태륵충일본주P23기인적핵감산동원성체99.4%,표체적융합단백대소약위46 ku;유도시궤이접충배양후2 h위최가,유도시간이6 h위최가,유도온도이34℃위최가,0.008～1.000 mmol/L적IPTC대표체량적영향불대.Western blotting검측표명해단백구유교호적항원성.[결론]위우슬씨태륵충병적면역학진단화예방등연구전정료기출.
[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti.[Method]A pair of specific primers was designed according to the sequence of P23 major surface protein of T.sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T.sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23.Positive clones were identified by PCR screening and restriction digestion.A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E.coil BL21.After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp.Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D64447).The expressed fusion protein was 46 ku in molecular mass.Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression.Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T,sergenti.
