检验医学与临床
檢驗醫學與臨床
검험의학여림상
JOURNAL OF LABORATORY MEDICINE AND CLINICAL SCIENCES
2014年
22期
3094-3096
,共3页
严子琴%刘丽%刘俊%施思%李海龙%蒋哲%孟凡国%周海梦%徐蓓%丁先锋%胡卫江%欧文斌
嚴子琴%劉麗%劉俊%施思%李海龍%蔣哲%孟凡國%週海夢%徐蓓%丁先鋒%鬍衛江%歐文斌
엄자금%류려%류준%시사%리해룡%장철%맹범국%주해몽%서배%정선봉%호위강%구문빈
甲胎蛋白%毕赤酵母%蛋白表达%蛋白纯化
甲胎蛋白%畢赤酵母%蛋白錶達%蛋白純化
갑태단백%필적효모%단백표체%단백순화
alpha-fetoprotein%Pichia%expression%purification
目的：从毕赤酵母GS115蛋白表达体系入手，实现人源甲胎蛋白（AFP）的重组克隆、异源高效表达、蛋白纯化并得到具有临床免疫原性的高纯度AFP。方法通过聚合酶链反应（PCR）从重组质粒pReceiver‐AFP扩增到目的基因片段，构建酵母重组表达质pPICZαA‐AFP，经测序比对与GenBank公开的AFP序列一致。将重组质粒pPICZαA‐AFP转入毕赤酵母GS115，通过甲醇诱导表达目的蛋白AFP。收集蛋白后用硫酸铵分级沉淀法对AFP进行纯化，高效液相色谱法（HPLC）检测纯度，在临床医院通过电化学发光法对其蛋白含量定值。结果研究发现AFP在酵母GS115中经甲醇诱导表达主要分泌到胞外培养基上清，免疫印迹试验能够检测到AFP蛋白条带，经硫酸铵分级沉淀，87％AFP目的蛋白存在于55％硫酸铵沉淀集份，HPLC检测其纯度为98．844％，电化学发光法检测其水平为3473ng／mL。最后与临床肝癌患者血清标本比对，蛋白的相对分子质量大小一致，表明其糖基化水平与临床标本相当。结论通过毕赤酵母甲醇诱导表达系统可以制备高纯度的、具有生物活性的、可用于肿瘤临床检测的AFP标准物质及其可以溯源的校准品、质控品和相关诊断试剂。
目的：從畢赤酵母GS115蛋白錶達體繫入手，實現人源甲胎蛋白（AFP）的重組剋隆、異源高效錶達、蛋白純化併得到具有臨床免疫原性的高純度AFP。方法通過聚閤酶鏈反應（PCR）從重組質粒pReceiver‐AFP擴增到目的基因片段，構建酵母重組錶達質pPICZαA‐AFP，經測序比對與GenBank公開的AFP序列一緻。將重組質粒pPICZαA‐AFP轉入畢赤酵母GS115，通過甲醇誘導錶達目的蛋白AFP。收集蛋白後用硫痠銨分級沉澱法對AFP進行純化，高效液相色譜法（HPLC）檢測純度，在臨床醫院通過電化學髮光法對其蛋白含量定值。結果研究髮現AFP在酵母GS115中經甲醇誘導錶達主要分泌到胞外培養基上清，免疫印跡試驗能夠檢測到AFP蛋白條帶，經硫痠銨分級沉澱，87％AFP目的蛋白存在于55％硫痠銨沉澱集份，HPLC檢測其純度為98．844％，電化學髮光法檢測其水平為3473ng／mL。最後與臨床肝癌患者血清標本比對，蛋白的相對分子質量大小一緻，錶明其糖基化水平與臨床標本相噹。結論通過畢赤酵母甲醇誘導錶達繫統可以製備高純度的、具有生物活性的、可用于腫瘤臨床檢測的AFP標準物質及其可以溯源的校準品、質控品和相關診斷試劑。
목적：종필적효모GS115단백표체체계입수，실현인원갑태단백（AFP）적중조극륭、이원고효표체、단백순화병득도구유림상면역원성적고순도AFP。방법통과취합매련반응（PCR）종중조질립pReceiver‐AFP확증도목적기인편단，구건효모중조표체질pPICZαA‐AFP，경측서비대여GenBank공개적AFP서렬일치。장중조질립pPICZαA‐AFP전입필적효모GS115，통과갑순유도표체목적단백AFP。수집단백후용류산안분급침정법대AFP진행순화，고효액상색보법（HPLC）검측순도，재림상의원통과전화학발광법대기단백함량정치。결과연구발현AFP재효모GS115중경갑순유도표체주요분비도포외배양기상청，면역인적시험능구검측도AFP단백조대，경류산안분급침정，87％AFP목적단백존재우55％류산안침정집빈，HPLC검측기순도위98．844％，전화학발광법검측기수평위3473ng／mL。최후여림상간암환자혈청표본비대，단백적상대분자질량대소일치，표명기당기화수평여림상표본상당。결론통과필적효모갑순유도표체계통가이제비고순도적、구유생물활성적、가용우종류림상검측적AFP표준물질급기가이소원적교준품、질공품화상관진단시제。
Objective To realize the recombination clone ,heterologic high‐efficiency expression and protein purification of human‐source alpha‐fetoprotein(AFP)and to obtain the high purity of AFP with clinical bioactivity by the pPICZaA pichia expression systems .Methods The human recombinant expression plasmid pPICZαA‐AFP was generated by PCR from pReceiver‐AFP ,which AFP sequence was in accordance with GenBank .The AFP protein was expressed by methanol induction in pichia GS115 ,and purified by ammonium sulfate precipitation .The expression and purification of AFP was evaluated by SDS‐PAGE ,western blotting and HPLC ,and the value of AFP was quantified by chemiluminescence immunoassay in clinical hospital .Results Recombinant human AFP expressed in the pichia GS115 was mainly secreted into the extracellular medium supernatant ,and also contained the immunogenicity .87% of AFP was in 55% ammonium sulfate precipitate after ammonium sulfate fractionation ,which purity was 98 .844% and value was 3 .473 ng/mL .Recombinant human AFP showed the same size of the protein molecular weight as the clini‐cal hepatocellular carcinoma(HCC)serum samples ,indicating that the glycosylation level of recombinant AFP was comparable with clinical samples .Conclusion The high purified recombinant human‐resouce AFP with biological ac‐tivity could be used to prepare AFP reference materials ,traceable calibrator ,controls and related diagnostic reagents by Pichia expression system ,which could be used for clinical tumor diagnosis and therapeutic detection .
