中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2015年
1期
77-81
,共5页
颗粒酶A%克隆%大肠杆菌%表达%活性测定
顆粒酶A%剋隆%大腸桿菌%錶達%活性測定
과립매A%극륭%대장간균%표체%활성측정
Granzyme A%Cloning%E.coli%Expression%Activity assay
目的：克隆和表达人颗粒酶A（ Granzyme A，GzmA）基因的活性片段（ active Granzyme A，aGzmA）并进行活性鉴定。方法：以全长人GzmA基因为模板，PCR扩增aGzmA基因片段，限制性内切酶NdeⅠ和XhoⅠ双酶切后插入原核表达载体pET24a（＋），将重组质粒转化大肠杆菌BL21（DE3），IPTG诱导表达，表达产物经SDS－PAGE和Western blot进行分析。重组蛋白用镍层析柱亲和层析进行纯化后，利用BLT底物溶液进行活性鉴定。结果：PCR扩增得到长约700 bp的基因片段。重组质粒pET24a－aGzmA经酶切和测序证实aGzmA基因序列正确插入载体质粒中。 SDS－PAGE显示在26 kD处有一特异性蛋白条带。 Western blot证实该蛋白可与小鼠抗His 单克隆抗体发生特异性结合。利用镍柱亲和层析法可从包涵体中纯化得到纯度较高的重组蛋白，且具有较好的酶活性。结论：成功制备了具有生物学活性的人颗粒酶A重组蛋白。
目的：剋隆和錶達人顆粒酶A（ Granzyme A，GzmA）基因的活性片段（ active Granzyme A，aGzmA）併進行活性鑒定。方法：以全長人GzmA基因為模闆，PCR擴增aGzmA基因片段，限製性內切酶NdeⅠ和XhoⅠ雙酶切後插入原覈錶達載體pET24a（＋），將重組質粒轉化大腸桿菌BL21（DE3），IPTG誘導錶達，錶達產物經SDS－PAGE和Western blot進行分析。重組蛋白用鎳層析柱親和層析進行純化後，利用BLT底物溶液進行活性鑒定。結果：PCR擴增得到長約700 bp的基因片段。重組質粒pET24a－aGzmA經酶切和測序證實aGzmA基因序列正確插入載體質粒中。 SDS－PAGE顯示在26 kD處有一特異性蛋白條帶。 Western blot證實該蛋白可與小鼠抗His 單剋隆抗體髮生特異性結閤。利用鎳柱親和層析法可從包涵體中純化得到純度較高的重組蛋白，且具有較好的酶活性。結論：成功製備瞭具有生物學活性的人顆粒酶A重組蛋白。
목적：극륭화표체인과립매A（ Granzyme A，GzmA）기인적활성편단（ active Granzyme A，aGzmA）병진행활성감정。방법：이전장인GzmA기인위모판，PCR확증aGzmA기인편단，한제성내절매NdeⅠ화XhoⅠ쌍매절후삽입원핵표체재체pET24a（＋），장중조질립전화대장간균BL21（DE3），IPTG유도표체，표체산물경SDS－PAGE화Western blot진행분석。중조단백용얼층석주친화층석진행순화후，이용BLT저물용액진행활성감정。결과：PCR확증득도장약700 bp적기인편단。중조질립pET24a－aGzmA경매절화측서증실aGzmA기인서렬정학삽입재체질립중。 SDS－PAGE현시재26 kD처유일특이성단백조대。 Western blot증실해단백가여소서항His 단극륭항체발생특이성결합。이용얼주친화층석법가종포함체중순화득도순도교고적중조단백，차구유교호적매활성。결론：성공제비료구유생물학활성적인과립매A중조단백。
Objective:To clone and express active domain of human granzyme A ( aGzmA ) and detect its biological activity.Methods:Human aGzmA gene was amplified by PCR from the full-length human granzyme A and inserted into prokaryotic ex-pression vector pET24a(+).The constructed recombinant plasmid pET24a-aGzmA was transformed to E.coli BL21(DE3) and induced with IPTG.The expressed product was identified by SDS-PAGE and Western blot.The recombinant protein was purified by the Ni2+affinity column chromatography and the enzyme activity was assayed with BLT substrate.Results: A DNA fragment of 700 bp was amplified by PCR.The recombinant plasmid pET24a-aGzmA identified by enzyme-digesting analysis and sequencing showed that aGzmA gene was inserted into vector plasmid correctly.SDS-PAGE analysis showed that there was a specific protein with a relative molecular mass of about 26 kD.Western blot analysis indicated that the protein could react with mouse anti-His monoclonal antibody specifically.The recombinant protein with high purity could be acquired from the inclusion bodies by the Ni2+ affinity column chromatography and the purified protein had good enzyme activity.Conclusion: The recombinant human granzyme A with good biological activity was prepared successfully.
