重組人單鏈IL-23的構(gòu)建、表達(dá)及生物學(xué)活性的鑒定
發(fā)布時(shí)間：2018-08-17 14:58
【摘要】：目的： (1)構(gòu)建重組人單鏈IL-23(hscIL-23)融合基因及pMD18-T-hscIL-23質(zhì)粒； (2)構(gòu)建原核表達(dá)質(zhì)粒pET-28a(+)-hscIL-23和真核表達(dá)質(zhì)粒pcDNA3.1(+)-hscIL-23； (3)原核表達(dá)hscIL-23重組蛋白； (4)真核表達(dá)hscIL-23重組蛋白； (5)初步鑒定真核表達(dá)的hscIL-23的生物學(xué)活性。 方法： (1)設(shè)計(jì)引物，應(yīng)用重疊延伸PCR技術(shù)通過一柔性接頭（linker）(Gly4Ser)3串聯(lián)擴(kuò)增自本實(shí)驗(yàn)室前期構(gòu)建的pMD18-T-p40質(zhì)粒和pMD18-T-p19質(zhì)粒的IL-23p40和p19亞基基因，使p40亞基基因在前，p19亞基基因在后，即p40+linker+p19（簡(jiǎn)寫為hscIL-23）。將hscIL-23融合基因插入至pMD18-T載體中，經(jīng)基因測(cè)序驗(yàn)證融合基因是否正確。 (2)提取pMD18-T-hscIL-23質(zhì)粒DNA，經(jīng)HindⅢ和Nhe I雙酶切，純化后的酶切產(chǎn)物hscIL-23基因通過T4連接酶與經(jīng)Hind Ⅲ, Nhe I雙酶切并純化的pET-28a(+)原核表達(dá)質(zhì)粒連接，構(gòu)建原核表達(dá)質(zhì)粒pET-28a(+)-hscIL-23，采用同樣的方法構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.1(+)-hscIL-23。 (3)將原核表達(dá)質(zhì)粒pET-28a(+)-hscIL-23轉(zhuǎn)化至大腸埃希菌BL21(DE3+)中，IPTG誘導(dǎo)表達(dá)帶有His標(biāo)簽的重組蛋白，采用鎳柱親和層析純化重組蛋白，，采用SDS-PAGE電泳和Western blot分析鑒定表達(dá)產(chǎn)物。 (4) pcDNA3.1(+)-hscIL-23質(zhì)粒按不同比例通過陽離子聚合物介導(dǎo)轉(zhuǎn)染HeLa細(xì)胞，同時(shí)設(shè)轉(zhuǎn)染pcDNA3.1(+)質(zhì)粒組和未轉(zhuǎn)染質(zhì)粒組，轉(zhuǎn)染48h后分別收集細(xì)胞及細(xì)胞培養(yǎng)上清。以人IL-23ELISA試劑盒鑒定各組細(xì)胞培養(yǎng)上清中hscIL-23融合蛋白的含量，通過與標(biāo)準(zhǔn)品比對(duì)，測(cè)定各組細(xì)胞培養(yǎng)上清中hscIL-23的表達(dá)水平。 (5)分離人PBMCs，加入收集的各組細(xì)胞培養(yǎng)上清及PHA-P共培養(yǎng)72h，MTT法檢測(cè)各組人淋巴細(xì)胞的增殖水平。 結(jié)果： (1)構(gòu)建的pMD18T-hscIL-23質(zhì)粒經(jīng)測(cè)序鑒定，插入片段序列序列與預(yù)期完全一致。構(gòu)建的pcDNA3.1(+)hscIL-23和pET-28a(+)-hscIL-23表達(dá)質(zhì)粒，其基因測(cè)序結(jié)果同樣無改變，p40、p19、linker的連接順序、方向及序列均與預(yù)期相符。 (2)原核表達(dá)質(zhì)粒pET-28a(+)-hscIL-23轉(zhuǎn)化的大腸桿菌可成功表達(dá)重組蛋白。經(jīng)SDS-PAGE和Western blot鑒定，所表達(dá)重組融合蛋白（hscIL-23）約為60kd，且能為hscIL-23p19抗體所識(shí)別。 (3)轉(zhuǎn)染各組質(zhì)粒的HeLa細(xì)胞中， ELISA檢測(cè)HeLa細(xì)胞在轉(zhuǎn)染質(zhì)粒48h后培養(yǎng)上清液中hscIL-23蛋白的含量，其中未轉(zhuǎn)染組細(xì)胞培養(yǎng)上清為9.11±11.78pg/ml，轉(zhuǎn)染pcDNA3.1(+)組的含量為10.47±9.04pg/ml，而轉(zhuǎn)染pcDNA3.1(+)-pscIL-12組為254.34±2.77pg/ml，顯著高于前兩組（P0.01）。 (4)通過MTT法檢測(cè)各組細(xì)胞培養(yǎng)上清刺激人淋巴細(xì)胞的增殖結(jié)果，未轉(zhuǎn)染質(zhì)粒組在570nm處的吸光度為0.553±0.038，PHA-P刺激組（陽性對(duì)照組）為1.968±0.018，轉(zhuǎn)染pcDNA3.1(+)組為0.595±0.289，轉(zhuǎn)染pcDNA3.1(+)-hscIL-23組組為1.135±0.048，顯著高于未轉(zhuǎn)染質(zhì)粒組和轉(zhuǎn)染pcDNA3.1(+)組（P0.01）。 結(jié)論： (1)成功構(gòu)建了原核表達(dá)質(zhì)粒pET-28a(+)-hscIL-23和真核表達(dá)質(zhì)粒pcDNA3.1(+)-hscIL-23； (2) pET-28a(+)-hscIL-23重組質(zhì)粒在大腸桿菌內(nèi)表達(dá)出目的蛋白； (3) pcDNA3.1(+)-hscIL-23在HeLa細(xì)胞中成功表達(dá)hscIL-23融合蛋白； (4)含真核表達(dá)hscIL-23蛋白的細(xì)胞培養(yǎng)上清能夠刺激人淋巴細(xì)胞增殖。
[Abstract]:Objective:
(1) to construct recombinant human single strand IL-23 (hscIL-23) fusion gene and pMD18-T-hscIL-23 plasmid.
(2) construction of prokaryotic expression plasmid pET-28a (+) -hscIL-23 and eukaryotic expression plasmid pcDNA3.1 (+) -hscIL-23;
(3) prokaryotic expression of hscIL-23 recombinant protein;
(4) eukaryotic expression of hscIL-23 recombinant protein.
(5) preliminary identification of the biological activity of eukaryotic expression of hscIL-23.
Method:
(1) Primers were designed to amplify the IL-23p40 and P19 subunits of pMD18-T-p40 plasmid and pMD18-T-p19 plasmid constructed in our laboratory by overlapping extension PCR through a flexible adapter (Gly4Ser) 3. The P40 subunit gene was pre-amplified and P19 subunit gene was post-amplified by overlapping extension PCR. Inserted into the pMD18-T vector, the gene was sequenced to verify whether the fusion gene was correct.
(2) The plasmid DNA of pMD18-T-hscIL-23 was extracted and digested by Hind I I I and Nhe I. The purified hscIL-23 gene was linked to the prokaryotic expression plasmid of pET-28a(+) digested and purified by Hind I I I and Nhe I. The prokaryotic expression plasmid pET-28a(+) -hscIL-23 was constructed by the same method. IL-23.
(3) Prokaryotic expression plasmid pET-28a(+) -hscIL-23 was transformed into Escherichia coli BL21 (DE3+). IPTG induced the expression of recombinant protein with His tag. The recombinant protein was purified by nickel column affinity chromatography and identified by SDS-PAGE electrophoresis and Western blot.
(4) pcDNA3.1 (+) - hscIL-23 plasmid was transfected into HeLa cells by cationic polymer in different proportions. At the same time, pcDNA3.1 (+) plasmid group and non-transfected plasmid group were set up. After 48 hours of transfection, the supernatant of cells and cell culture were collected. The content of hscIL-23 fusion protein in the supernatant of each group was identified by human IL-23 ELISA kit. The expression level of hscIL-23 in cell culture supernatants was determined by standard comparison.
(5) Human PBMCs were isolated and cultured in the supernatant and PHA-P for 72 hours. MTT assay was used to detect the proliferation of human lymphocytes in each group.
Result:
(1) The constructed plasmid pMD18T-hscIL-23 was sequenced and identified, and the sequence of the inserted fragment was identical with the expected sequence.
(2) Escherichia coli transformed with prokaryotic expression plasmid pET-28a(+) -hscIL-23 could successfully express the recombinant protein. The recombinant fusion protein (hscIL-23) was identified by SDS-PAGE and Western blot and could be recognized by hscIL-23p19 antibody.
(3) In HeLa cells transfected with plasmids, the content of hscIL-23 protein in culture supernatant of HeLa cells 48 hours after transfection was detected by ELISA. The cell culture supernatant of untransfected group was 9.11 (11.78) pg/ml, that of pcDNA3.1 (+) transfected group was 10.47 (9.04) pg/ml, and that of pcDNA3.1 (+) -pscIL-12 transfected group was 254.34 (2.77) pg/ml, which was significantly higher than that of the former two groups. Group (P0.01).
(4) MTT assay was used to detect the proliferation of human lymphocytes stimulated by the culture supernatant of each group. The absorbance of the untransfected plasmid group at 570 nm was 0.553.038, the PHA-P stimulation group (positive control group) was 1.968.018, the pcDNA3.1 (+) transfected group was 0.595.289, and the pcDNA3.1 (+) - hscIL-23 transfected group was 1.135.048, which was significantly higher than that of the untransfected plasmid group Group and transfected pcDNA3.1 (+) group (P0.01).
Conclusion:
(1) the prokaryotic expression plasmid pET-28a (+) -hscIL-23 and eukaryotic expression plasmid pcDNA3.1 (+) -hscIL-23 were successfully constructed.
(2) pET-28a (+) -hscIL-23 recombinant plasmid expressed the target protein in E. coli.
(3) pcDNA3.1 (+) -hscIL-23 successfully expressed hscIL-23 fusion protein in HeLa cells.
(4) cell culture supernatant containing eukaryotic expression of hscIL-23 protein can stimulate the proliferation of human lymphocytes.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1
本文編號(hào)：2188010
[Abstract]:Objective:
(1) to construct recombinant human single strand IL-23 (hscIL-23) fusion gene and pMD18-T-hscIL-23 plasmid.
(2) construction of prokaryotic expression plasmid pET-28a (+) -hscIL-23 and eukaryotic expression plasmid pcDNA3.1 (+) -hscIL-23;
(3) prokaryotic expression of hscIL-23 recombinant protein;
(4) eukaryotic expression of hscIL-23 recombinant protein.
(5) preliminary identification of the biological activity of eukaryotic expression of hscIL-23.
Method:
(1) Primers were designed to amplify the IL-23p40 and P19 subunits of pMD18-T-p40 plasmid and pMD18-T-p19 plasmid constructed in our laboratory by overlapping extension PCR through a flexible adapter (Gly4Ser) 3. The P40 subunit gene was pre-amplified and P19 subunit gene was post-amplified by overlapping extension PCR. Inserted into the pMD18-T vector, the gene was sequenced to verify whether the fusion gene was correct.
(2) The plasmid DNA of pMD18-T-hscIL-23 was extracted and digested by Hind I I I and Nhe I. The purified hscIL-23 gene was linked to the prokaryotic expression plasmid of pET-28a(+) digested and purified by Hind I I I and Nhe I. The prokaryotic expression plasmid pET-28a(+) -hscIL-23 was constructed by the same method. IL-23.
(3) Prokaryotic expression plasmid pET-28a(+) -hscIL-23 was transformed into Escherichia coli BL21 (DE3+). IPTG induced the expression of recombinant protein with His tag. The recombinant protein was purified by nickel column affinity chromatography and identified by SDS-PAGE electrophoresis and Western blot.
(4) pcDNA3.1 (+) - hscIL-23 plasmid was transfected into HeLa cells by cationic polymer in different proportions. At the same time, pcDNA3.1 (+) plasmid group and non-transfected plasmid group were set up. After 48 hours of transfection, the supernatant of cells and cell culture were collected. The content of hscIL-23 fusion protein in the supernatant of each group was identified by human IL-23 ELISA kit. The expression level of hscIL-23 in cell culture supernatants was determined by standard comparison.
(5) Human PBMCs were isolated and cultured in the supernatant and PHA-P for 72 hours. MTT assay was used to detect the proliferation of human lymphocytes in each group.
Result:
(1) The constructed plasmid pMD18T-hscIL-23 was sequenced and identified, and the sequence of the inserted fragment was identical with the expected sequence.
(2) Escherichia coli transformed with prokaryotic expression plasmid pET-28a(+) -hscIL-23 could successfully express the recombinant protein. The recombinant fusion protein (hscIL-23) was identified by SDS-PAGE and Western blot and could be recognized by hscIL-23p19 antibody.
(3) In HeLa cells transfected with plasmids, the content of hscIL-23 protein in culture supernatant of HeLa cells 48 hours after transfection was detected by ELISA. The cell culture supernatant of untransfected group was 9.11 (11.78) pg/ml, that of pcDNA3.1 (+) transfected group was 10.47 (9.04) pg/ml, and that of pcDNA3.1 (+) -pscIL-12 transfected group was 254.34 (2.77) pg/ml, which was significantly higher than that of the former two groups. Group (P0.01).
(4) MTT assay was used to detect the proliferation of human lymphocytes stimulated by the culture supernatant of each group. The absorbance of the untransfected plasmid group at 570 nm was 0.553.038, the PHA-P stimulation group (positive control group) was 1.968.018, the pcDNA3.1 (+) transfected group was 0.595.289, and the pcDNA3.1 (+) - hscIL-23 transfected group was 1.135.048, which was significantly higher than that of the untransfected plasmid group Group and transfected pcDNA3.1 (+) group (P0.01).
Conclusion:
(1) the prokaryotic expression plasmid pET-28a (+) -hscIL-23 and eukaryotic expression plasmid pcDNA3.1 (+) -hscIL-23 were successfully constructed.
(2) pET-28a (+) -hscIL-23 recombinant plasmid expressed the target protein in E. coli.
(3) pcDNA3.1 (+) -hscIL-23 successfully expressed hscIL-23 fusion protein in HeLa cells.
(4) cell culture supernatant containing eukaryotic expression of hscIL-23 protein can stimulate the proliferation of human lymphocytes.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 徐國(guó)榮;程娜;張倫理;;結(jié)核性腦膜炎IL-23的測(cè)定及意義[J];廣東醫(yī)學(xué);2010年20期
2 白艷梅;楊潔;李杰;李燕;王瑞倉;王超;袁軍;王素云;郝洪嶺;;IL-23單獨(dú)或聯(lián)合IL-2對(duì)人PBMC增殖及抗白血病活性影響的研究[J];免疫學(xué)雜志;2011年12期
3 鄭紫丹;萬曉強(qiáng);劉梁英;;潰瘍性結(jié)腸炎患者血清IL-23和IL-17的水平變化及意義[J];細(xì)胞與分子免疫學(xué)雜志;2011年02期
4 吳碧蓮;陳少強(qiáng);賈小力;;梭華-Sofast介導(dǎo)綠色熒光蛋白轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞的轉(zhuǎn)染效果[J];中國(guó)組織工程研究與臨床康復(fù);2008年16期
5 楊晨濤;魏林;曾瑞紅;;IL-23、IL-27對(duì)RSV重組蛋白疫苗免疫原性的影響[J];中國(guó)免疫學(xué)雜志;2011年03期
6 楊帆;伍偉鋒;顏玉鸞;龐羽;孔清;;外源性IL-23對(duì)病毒性心肌炎小鼠Th17細(xì)胞增殖的影響[J];中國(guó)免疫學(xué)雜志;2011年11期
本文編號(hào)：2188010
本文鏈接：http://sikaile.net/xiyixuelunwen/2188010.html
最近更新
教材專著
