中国人兽共患病学报
中國人獸共患病學報
중국인수공환병학보
CHINESE JOURNAL OF ZOONOSES
2012年
9期
861-868
,共8页
张超颖%郭果为%王正朝%黄晓红
張超穎%郭果為%王正朝%黃曉紅
장초영%곽과위%왕정조%황효홍
长角血蜱%谷胱甘肽过氧化物酶%生物信息学分析%克隆%定点突变%原核表达%Western印迹
長角血蜱%穀胱甘肽過氧化物酶%生物信息學分析%剋隆%定點突變%原覈錶達%Western印跡
장각혈비%곡광감태과양화물매%생물신식학분석%극륭%정점돌변%원핵표체%Western인적
Haemaphysalis longicornis%glutathione peroxidase%cloning%site-directed mutagenesis%prokaryotic expression
目的 长角血蜱(Hemaphysalis longicornis)不仅吸食家畜血甚至人血,还是多种疾病的传播媒介,对该蜱的研究具有重要的医学和兽医学意义.在大肠杆菌中表达长角血蜱的谷胱甘肽过氧化物酶(glutathione peroxidase)(HlGPx),为该酶在长角血蜱中的生理作用研究奠定基础,为该病媒的免疫防控提供参考.方法 根据长角血蜱EST文库提示的片段序列的信息,设计引物采用PCR方法从长角血蜱成蜱的cDNA中扩增谷胱甘肽过氧化物酶基因.利用定点突变将该基因编码序列中一个编码硒半胱氨酸的密码子TGA突变为通用半胱氨酸密码子TGC,与表达载体pGEX-4T-1重组,转化BL21(DE3)pLysS,IPTG诱导表达,SDS-PAGE及免疫印迹分析.结果 成功获得了长角血蜱谷胱甘肽过氧化物酶全长序列,突变后在大肠杆菌中诱导表达了约51kDa的重组蛋白,Western-blot显示表达产物与抗GST抗体有很强的交叉反应.结论 突变的HlGPx可以在大肠杆菌中表达并可用于下一步制备免疫血清及功能分析.
目的 長角血蜱(Hemaphysalis longicornis)不僅吸食傢畜血甚至人血,還是多種疾病的傳播媒介,對該蜱的研究具有重要的醫學和獸醫學意義.在大腸桿菌中錶達長角血蜱的穀胱甘肽過氧化物酶(glutathione peroxidase)(HlGPx),為該酶在長角血蜱中的生理作用研究奠定基礎,為該病媒的免疫防控提供參攷.方法 根據長角血蜱EST文庫提示的片段序列的信息,設計引物採用PCR方法從長角血蜱成蜱的cDNA中擴增穀胱甘肽過氧化物酶基因.利用定點突變將該基因編碼序列中一箇編碼硒半胱氨痠的密碼子TGA突變為通用半胱氨痠密碼子TGC,與錶達載體pGEX-4T-1重組,轉化BL21(DE3)pLysS,IPTG誘導錶達,SDS-PAGE及免疫印跡分析.結果 成功穫得瞭長角血蜱穀胱甘肽過氧化物酶全長序列,突變後在大腸桿菌中誘導錶達瞭約51kDa的重組蛋白,Western-blot顯示錶達產物與抗GST抗體有很彊的交扠反應.結論 突變的HlGPx可以在大腸桿菌中錶達併可用于下一步製備免疫血清及功能分析.
목적 장각혈비(Hemaphysalis longicornis)불부흡식가축혈심지인혈,환시다충질병적전파매개,대해비적연구구유중요적의학화수의학의의.재대장간균중표체장각혈비적곡광감태과양화물매(glutathione peroxidase)(HlGPx),위해매재장각혈비중적생리작용연구전정기출,위해병매적면역방공제공삼고.방법 근거장각혈비EST문고제시적편단서렬적신식,설계인물채용PCR방법종장각혈비성비적cDNA중확증곡광감태과양화물매기인.이용정점돌변장해기인편마서렬중일개편마서반광안산적밀마자TGA돌변위통용반광안산밀마자TGC,여표체재체pGEX-4T-1중조,전화BL21(DE3)pLysS,IPTG유도표체,SDS-PAGE급면역인적분석.결과 성공획득료장각혈비곡광감태과양화물매전장서렬,돌변후재대장간균중유도표체료약51kDa적중조단백,Western-blot현시표체산물여항GST항체유흔강적교차반응.결론 돌변적HlGPx가이재대장간균중표체병가용우하일보제비면역혈청급공능분석.
Ticks transmit various diseases to livestock and human beings. The researches on ticks are of great importance in medical and veterinary sciences. To express a glutathione peroxidase gene (HlGPx) from Hemaphysalis longicornis in Escherichia coli (E. coli) so as to provide basis for further studies, the gene was amplified from cDNAs of H. longicornis adult ticks by PCR prompted by information from EST library. The TGA codon encoding selenocysteine in the gene was mutated into the universal genetic code for cysteine,TGC, by site-directed mutagenesis. E. coli was transformed with the recombinant expression vector pGEX-4T-1/HlGPx'(the mutated HlGPx) and induced to express recombinant HlGPx', which was then analyzed by sodium dodecylsulphate-polyacrylamide gel electrophoresis and Western blotting. The result showed that the complete coding sequence of HlGPx was obtained successfully, the deduced polypeptide was 223 aa, with a calculated molecular mass of 25.0 kDa; the recombinant fusion protein was approximately 51 kDa, correspondent to the calculated. The antibody against glutathione S-transferase (GST) recognized the recombinant protein fused with GST in a Western blotting assay, which confirmed the result above-mentioned. In conclusion, the mutated HlGPx was expressed in E. coli successfully and it could be used for further preparation of immune serum and functional analysis.
