中国生化药物杂志
中國生化藥物雜誌
중국생화약물잡지
CHINESE JOURNAL OF BIOCHEMICAL PHARMACEUTICS
2015年
7期
1-4
,共4页
B细胞激活因子%cDNA克隆%原核表达%蛋白纯化
B細胞激活因子%cDNA剋隆%原覈錶達%蛋白純化
B세포격활인자%cDNA극륭%원핵표체%단백순화
B cell activating factor%cDNA cloning%prokaryotic expression%protein purification
目的：本研究对TNF家族B细胞激活因子（ B cell activating factor to the TNF family， BAFF）的胞外区134～285位氨基酸残基进行克隆，构建原核表达载体pET21a－sBAFF，并进行表达及纯化。方法以K562细胞系的cDNA为模板，采用PCR扩增sBAFF基因，构建pET21a－sBAFF。在大肠杆菌BL21（DE3）中经IPTG诱导表达sBAFF，超声碎菌，提取包涵体，经Ni2＋－IDA亲和层析纯化，复性，采用SDS－PAGE鉴定纯化产物。结果通过优化确定了目的蛋白适宜的诱导温度和诱导时间。表达的sBAFF相对分子量约为18000，目的蛋白占菌体总蛋白的38．59％，且主要以包涵体的形式存在。经过蛋白复性后有38．14％的sBAFF聚合成了有活性的三聚体，代表错误折叠的二聚体含量极低。结论建立了成功的复性工艺，目的蛋白经过复性后形成具有活性的三聚体，为进一步研究BAFF的生物学功能以及以BAFF为靶点的自身免疫疾病药物开发奠定了基础。
目的：本研究對TNF傢族B細胞激活因子（ B cell activating factor to the TNF family， BAFF）的胞外區134～285位氨基痠殘基進行剋隆，構建原覈錶達載體pET21a－sBAFF，併進行錶達及純化。方法以K562細胞繫的cDNA為模闆，採用PCR擴增sBAFF基因，構建pET21a－sBAFF。在大腸桿菌BL21（DE3）中經IPTG誘導錶達sBAFF，超聲碎菌，提取包涵體，經Ni2＋－IDA親和層析純化，複性，採用SDS－PAGE鑒定純化產物。結果通過優化確定瞭目的蛋白適宜的誘導溫度和誘導時間。錶達的sBAFF相對分子量約為18000，目的蛋白佔菌體總蛋白的38．59％，且主要以包涵體的形式存在。經過蛋白複性後有38．14％的sBAFF聚閤成瞭有活性的三聚體，代錶錯誤摺疊的二聚體含量極低。結論建立瞭成功的複性工藝，目的蛋白經過複性後形成具有活性的三聚體，為進一步研究BAFF的生物學功能以及以BAFF為靶點的自身免疫疾病藥物開髮奠定瞭基礎。
목적：본연구대TNF가족B세포격활인자（ B cell activating factor to the TNF family， BAFF）적포외구134～285위안기산잔기진행극륭，구건원핵표체재체pET21a－sBAFF，병진행표체급순화。방법이K562세포계적cDNA위모판，채용PCR확증sBAFF기인，구건pET21a－sBAFF。재대장간균BL21（DE3）중경IPTG유도표체sBAFF，초성쇄균，제취포함체，경Ni2＋－IDA친화층석순화，복성，채용SDS－PAGE감정순화산물。결과통과우화학정료목적단백괄의적유도온도화유도시간。표체적sBAFF상대분자량약위18000，목적단백점균체총단백적38．59％，차주요이포함체적형식존재。경과단백복성후유38．14％적sBAFF취합성료유활성적삼취체，대표착오절첩적이취체함량겁저。결론건립료성공적복성공예，목적단백경과복성후형성구유활성적삼취체，위진일보연구BAFF적생물학공능이급이BAFF위파점적자신면역질병약물개발전정료기출。
Objective To construct pET21a-sBAFF by cloning the extracellular regions of 134-285 amino acids of BAFF, a member of human TNF family, and then express the gene in prokaryotic cells and purify the expressed product.Methods cDNA of K562 cell line was used as the template to amplify sBAFF gene to construct pET21a-sBAFF.Expression of sBAFF in E.coli BL21 was induced by IPTG, and the expressed proteins were assayed by SDS-PAGE.The bacteria were analyzed by sonication, and the target proteins mainly existed as inclusion bodies.Then sBAFF was purified by Ni2 +-IDA affinity chromatography.SDS-PAGE electrophoreses displayed that the expressed sBAFF migrated with a relative molecular weight of 18000.ResuIts The induction parameters such as temperature and inducing time were optimized.The target protein accounted for 38.59%of the total bacterial proteins.After refolding, 38.14% of sBAFF proteins were polymerized as an active trimer.The dimer form of sBAFF, which is representative of wrongly refolded product, accounted for very few.ConcIusion The expression and purification of BAFF which formed active trimer after refolding pave the way for its further function study and provide a novel approach for the development of BAFF-targeted therapeutics for autoimmune diseases.
