重組人單鏈IL-23的構建、表達及生物學活性的鑒定
發(fā)布時間：2018-08-17 14:58
【摘要】：目的： (1)構建重組人單鏈IL-23(hscIL-23)融合基因及pMD18-T-hscIL-23質粒； (2)構建原核表達質粒pET-28a(+)-hscIL-23和真核表達質粒pcDNA3.1(+)-hscIL-23； (3)原核表達hscIL-23重組蛋白； (4)真核表達hscIL-23重組蛋白； (5)初步鑒定真核表達的hscIL-23的生物學活性。 方法： (1)設計引物，應用重疊延伸PCR技術通過一柔性接頭（linker）(Gly4Ser)3串聯(lián)擴增自本實驗室前期構建的pMD18-T-p40質粒和pMD18-T-p19質粒的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（簡寫為hscIL-23）。將hscIL-23融合基因插入至pMD18-T載體中，經基因測序驗證融合基因是否正確。 (2)提取pMD18-T-hscIL-23質粒DNA，經HindⅢ和Nhe I雙酶切，純化后的酶切產物hscIL-23基因通過T4連接酶與經Hind Ⅲ, Nhe I雙酶切并純化的pET-28a(+)原核表達質粒連接，構建原核表達質粒pET-28a(+)-hscIL-23，采用同樣的方法構建真核表達質粒pcDNA3.1(+)-hscIL-23。 (3)將原核表達質粒pET-28a(+)-hscIL-23轉化至大腸埃希菌BL21(DE3+)中，IPTG誘導表達帶有His標簽的重組蛋白，采用鎳柱親和層析純化重組蛋白，，采用SDS-PAGE電泳和Western blot分析鑒定表達產物。 (4) pcDNA3.1(+)-hscIL-23質粒按不同比例通過陽離子聚合物介導轉染HeLa細胞，同時設轉染pcDNA3.1(+)質粒組和未轉染質粒組，轉染48h后分別收集細胞及細胞培養(yǎng)上清。以人IL-23ELISA試劑盒鑒定各組細胞培養(yǎng)上清中hscIL-23融合蛋白的含量，通過與標準品比對，測定各組細胞培養(yǎng)上清中hscIL-23的表達水平。 (5)分離人PBMCs，加入收集的各組細胞培養(yǎng)上清及PHA-P共培養(yǎng)72h，MTT法檢測各組人淋巴細胞的增殖水平。 結果： (1)構建的pMD18T-hscIL-23質粒經測序鑒定，插入片段序列序列與預期完全一致。構建的pcDNA3.1(+)hscIL-23和pET-28a(+)-hscIL-23表達質粒，其基因測序結果同樣無改變，p40、p19、linker的連接順序、方向及序列均與預期相符。 (2)原核表達質粒pET-28a(+)-hscIL-23轉化的大腸桿菌可成功表達重組蛋白。經SDS-PAGE和Western blot鑒定，所表達重組融合蛋白（hscIL-23）約為60kd，且能為hscIL-23p19抗體所識別。 (3)轉染各組質粒的HeLa細胞中， ELISA檢測HeLa細胞在轉染質粒48h后培養(yǎng)上清液中hscIL-23蛋白的含量，其中未轉染組細胞培養(yǎng)上清為9.11±11.78pg/ml，轉染pcDNA3.1(+)組的含量為10.47±9.04pg/ml，而轉染pcDNA3.1(+)-pscIL-12組為254.34±2.77pg/ml，顯著高于前兩組（P0.01）。 (4)通過MTT法檢測各組細胞培養(yǎng)上清刺激人淋巴細胞的增殖結果，未轉染質粒組在570nm處的吸光度為0.553±0.038，PHA-P刺激組（陽性對照組）為1.968±0.018，轉染pcDNA3.1(+)組為0.595±0.289，轉染pcDNA3.1(+)-hscIL-23組組為1.135±0.048，顯著高于未轉染質粒組和轉染pcDNA3.1(+)組（P0.01）。 結論： (1)成功構建了原核表達質粒pET-28a(+)-hscIL-23和真核表達質粒pcDNA3.1(+)-hscIL-23； (2) pET-28a(+)-hscIL-23重組質粒在大腸桿菌內表達出目的蛋白； (3) pcDNA3.1(+)-hscIL-23在HeLa細胞中成功表達hscIL-23融合蛋白； (4)含真核表達hscIL-23蛋白的細胞培養(yǎng)上清能夠刺激人淋巴細胞增殖。
[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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392.1
本文編號：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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392.1
【參考文獻】
相關期刊論文 前6條
1 徐國榮;程娜;張倫理;;結核性腦膜炎IL-23的測定及意義[J];廣東醫(yī)學;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單獨或聯(lián)合IL-2對人PBMC增殖及抗白血病活性影響的研究[J];免疫學雜志;2011年12期
3 鄭紫丹;萬曉強;劉梁英;;潰瘍性結腸炎患者血清IL-23和IL-17的水平變化及意義[J];細胞與分子免疫學雜志;2011年02期
4 吳碧蓮;陳少強;賈小力;;梭華-Sofast介導綠色熒光蛋白轉染骨髓間充質干細胞的轉染效果[J];中國組織工程研究與臨床康復;2008年16期
5 楊晨濤;魏林;曾瑞紅;;IL-23、IL-27對RSV重組蛋白疫苗免疫原性的影響[J];中國免疫學雜志;2011年03期
6 楊帆;伍偉鋒;顏玉鸞;龐羽;孔清;;外源性IL-23對病毒性心肌炎小鼠Th17細胞增殖的影響[J];中國免疫學雜志;2011年11期
本文編號：2188010
本文鏈接：http://sikaile.net/xiyixuelunwen/2188010.html
最近更新
教材專著
