人脂聯(lián)素球狀結(jié)構(gòu)域基因的真核、原核表達及多克隆抗體的制備
發(fā)布時間：2019-06-18 19:14
【摘要】：研究目的 利用桿狀病毒表達系統(tǒng)表達出有生物學(xué)活性的人脂聯(lián)素球狀結(jié)構(gòu)域(globular domain of adiponectin,gAd)蛋白,為研究gAd蛋白在糖尿病、心血管疾病、子癇等疾病中的作用機制奠定基礎(chǔ)。構(gòu)建gAd基因的原核表達載體,并經(jīng)誘導(dǎo)表達、純化gAd后,制備gAd多克隆抗體,為后續(xù)對人血清脂聯(lián)素檢測方法的建立奠定基礎(chǔ)。 研究方法 (1)第一部分:以人基因組為模板,PCR法擴增人gAd基因,將gAd基因與質(zhì)粒pFastBacHTB連接,轉(zhuǎn)化含有穿梭載體Bacmid的大腸桿菌DH10Bac。篩選轉(zhuǎn)座成功的重組穿梭載體Bacmid-gAd,通過脂質(zhì)體介導(dǎo),轉(zhuǎn)染昆蟲細胞Sf9,經(jīng)SDS-PAGE,免疫印跡法檢測表達產(chǎn)物。 (2)第二部分:以人基因組為模板,用PCR方法擴增人gAd基因,構(gòu)建原核表達載體PET-28a(+)-gAd,轉(zhuǎn)化大腸桿菌BL21(DE3),IPTG誘導(dǎo)蛋白表達,經(jīng)SDS-PAGE,免疫印跡法檢測并鑒定表達產(chǎn)物,表達的目的蛋白用鎳親和層析柱純化后免疫新西蘭大白兔,制備多克隆抗體。 研究結(jié)果 (1)利用真核桿狀病毒表達系統(tǒng)后,重組桿狀病毒感染的Sf9細胞形態(tài)變化明顯,SDS-PAGE圖譜,在相對分子質(zhì)量15～25KD之間有一條新生蛋白帶,免疫印跡法證實能與相應(yīng)的抗His標簽抗體結(jié)合。 (2)原核表達載體PET-28a(+)-gAd構(gòu)建成功,并獲得gAd重組蛋白,免疫印跡證實能與抗his標簽檢測抗體結(jié)合,重組蛋白經(jīng)純化后電泳條帶單一,制備的多克隆抗體經(jīng)間接ELISA法測得效價為1:32 000。免疫印跡證實,抗血清能特異性地識別gAd蛋白和胎盤組織中的脂聯(lián)素。 結(jié)論 (1)人gAd基因成功在昆蟲細胞中表達,為后續(xù)進一步研究脂聯(lián)素在糖尿病、心血管等疾病發(fā)病過程中的作用機制及治療價值奠定了基礎(chǔ)。 (2)成功構(gòu)建原核表達載體PET-28a(+)-gAd,并表達、純化重組蛋白gAd,制備的多克隆抗體特異性好,效價較高,為進一步制備針對gAd的單克隆抗體、進而建立檢測脂聯(lián)素的方法奠定了基礎(chǔ)。
[Abstract]:Objective to express human adiponectin spherical domain (globular domain of adiponectin,gAd protein with biological activity by baculovirus expression system, and to lay a foundation for studying the mechanism of gAd protein in diabetes, cardiovascular disease, eclampsia and other diseases. The prokaryotic expression vector of gAd gene was constructed and expressed by induction. after purification of gAd, gAd polyclonal antibody was prepared, which laid a foundation for the subsequent establishment of human serum adiponectin detection method. Methods (1) the first part: using the human genome as template, the human gAd gene was amplified by PCR. The gAd gene was ligated with plasmid pFastBacHTB and transformed into E. coli DH10Bac. containing shuttle vector Bacmid. The recombinant shuttle vector Bacmid-gAd, which was successfully inserted into insect cells, was mediated by liposomes and transformed into insect cells. The expressed products were detected by SDS-PAGE, immunoblotting. (2) the second part: using the human genome as template, the human gAd gene was amplified by PCR, and the prokaryotic expression vector PET-28a ()-gAd, was constructed to transform the expression of E. coli BL21 (DE3), IPTG induced protein). The expression product was detected and identified by SDS-PAGE, immunoblotting. The expressed target protein was purified by nickel affinity chromatography and immunized with New Zealand white rabbit to prepare polyclonal antibody. Results (1) after using eukaryotic baculovirus expression system, the morphology of Sf9 cells infected with recombinant baculovirus changed obviously, SDS-PAGE map, and there was a new protein band between the relative molecular weight 15~25KD. Immunoblotting confirmed that it could bind to the corresponding anti-His label antibody. (2) the prokaryotic expression vector PET-28a ()-gAd was successfully constructed and the recombinant protein of gAd was obtained. The recombinant protein could bind to the antibody detected by anti-his label by immunoblotting. After purification, the electrophoresis band of the recombinant protein was single, and the titer of the prepared polyclonal antibody was 1:32 000 by indirect ELISA method. Western imprinting confirmed that antiserum could specifically recognize gAd protein and adiponectin in placental tissue. Conclusion (1) Human gAd gene is successfully expressed in insect cells, which lays a foundation for further study on the mechanism and therapeutic value of adiponectin in the pathogenesis of diabetes, cardiovascular and other diseases. (2) the prokaryotic expression vector PET-28a ()-gAd, was successfully constructed and expressed. The polyclonal antibody prepared by purified recombinant protein gAd, had good specificity and high titer, which laid a foundation for the further preparation of monoclonal antibody against gAd and the establishment of a method for the detection of adiponectin.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341
本文編號：2501722
[Abstract]:Objective to express human adiponectin spherical domain (globular domain of adiponectin,gAd protein with biological activity by baculovirus expression system, and to lay a foundation for studying the mechanism of gAd protein in diabetes, cardiovascular disease, eclampsia and other diseases. The prokaryotic expression vector of gAd gene was constructed and expressed by induction. after purification of gAd, gAd polyclonal antibody was prepared, which laid a foundation for the subsequent establishment of human serum adiponectin detection method. Methods (1) the first part: using the human genome as template, the human gAd gene was amplified by PCR. The gAd gene was ligated with plasmid pFastBacHTB and transformed into E. coli DH10Bac. containing shuttle vector Bacmid. The recombinant shuttle vector Bacmid-gAd, which was successfully inserted into insect cells, was mediated by liposomes and transformed into insect cells. The expressed products were detected by SDS-PAGE, immunoblotting. (2) the second part: using the human genome as template, the human gAd gene was amplified by PCR, and the prokaryotic expression vector PET-28a ()-gAd, was constructed to transform the expression of E. coli BL21 (DE3), IPTG induced protein). The expression product was detected and identified by SDS-PAGE, immunoblotting. The expressed target protein was purified by nickel affinity chromatography and immunized with New Zealand white rabbit to prepare polyclonal antibody. Results (1) after using eukaryotic baculovirus expression system, the morphology of Sf9 cells infected with recombinant baculovirus changed obviously, SDS-PAGE map, and there was a new protein band between the relative molecular weight 15~25KD. Immunoblotting confirmed that it could bind to the corresponding anti-His label antibody. (2) the prokaryotic expression vector PET-28a ()-gAd was successfully constructed and the recombinant protein of gAd was obtained. The recombinant protein could bind to the antibody detected by anti-his label by immunoblotting. After purification, the electrophoresis band of the recombinant protein was single, and the titer of the prepared polyclonal antibody was 1:32 000 by indirect ELISA method. Western imprinting confirmed that antiserum could specifically recognize gAd protein and adiponectin in placental tissue. Conclusion (1) Human gAd gene is successfully expressed in insect cells, which lays a foundation for further study on the mechanism and therapeutic value of adiponectin in the pathogenesis of diabetes, cardiovascular and other diseases. (2) the prokaryotic expression vector PET-28a ()-gAd, was successfully constructed and expressed. The polyclonal antibody prepared by purified recombinant protein gAd, had good specificity and high titer, which laid a foundation for the further preparation of monoclonal antibody against gAd and the establishment of a method for the detection of adiponectin.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341
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