重组人单链IL-23的构建、表达及生物学活性的鉴定

发布时间：2018-08-17 14:58

【摘要】：目的： (1)构建重组人单链IL-23(hscIL-23)融合基因及pMD18-T-hscIL-23质粒； (2)构建原核表达质粒pET-28a(+)-hscIL-23和真核表达质粒pcDNA3.1(+)-hscIL-23； (3)原核表达hscIL-23重组蛋白； (4)真核表达hscIL-23重组蛋白； (5)初步鉴定真核表达的hscIL-23的生物学活性。方法： (1)设计引物，应用重叠延伸PCR技术通过一柔性接头（linker）(Gly4Ser)3串联扩增自本实验室前期构建的pMD18-T-p40质粒和pMD18-T-p19质粒的IL-23p40和p19亚基基因，使p40亚基基因在前，p19亚基基因在后，即p40+linker+p19（简写为hscIL-23）。将hscIL-23融合基因插入至pMD18-T载体中，经基因测序验证融合基因是否正确。 (2)提取pMD18-T-hscIL-23质粒DNA，经HindⅢ和Nhe I双酶切，纯化后的酶切产物hscIL-23基因通过T4连接酶与经Hind Ⅲ, Nhe I双酶切并纯化的pET-28a(+)原核表达质粒连接，构建原核表达质粒pET-28a(+)-hscIL-23，采用同样的方法构建真核表达质粒pcDNA3.1(+)-hscIL-23。 (3)将原核表达质粒pET-28a(+)-hscIL-23转化至大肠埃希菌BL21(DE3+)中，IPTG诱导表达带有His标签的重组蛋白，采用镍柱亲和层析纯化重组蛋白，，采用SDS-PAGE电泳和Western blot分析鉴定表达产物。 (4) pcDNA3.1(+)-hscIL-23质粒按不同比例通过阳离子聚合物介导转染HeLa细胞，同时设转染pcDNA3.1(+)质粒组和未转染质粒组，转染48h后分别收集细胞及细胞培养上清。以人IL-23ELISA试剂盒鉴定各组细胞培养上清中hscIL-23融合蛋白的含量，通过与标准品比对，测定各组细胞培养上清中hscIL-23的表达水平。 (5)分离人PBMCs，加入收集的各组细胞培养上清及PHA-P共培养72h，MTT法检测各组人淋巴细胞的增殖水平。结果： (1)构建的pMD18T-hscIL-23质粒经测序鉴定，插入片段序列序列与预期完全一致。构建的pcDNA3.1(+)hscIL-23和pET-28a(+)-hscIL-23表达质粒，其基因测序结果同样无改变，p40、p19、linker的连接顺序、方向及序列均与预期相符。 (2)原核表达质粒pET-28a(+)-hscIL-23转化的大肠杆菌可成功表达重组蛋白。经SDS-PAGE和Western blot鉴定，所表达重组融合蛋白（hscIL-23）约为60kd，且能为hscIL-23p19抗体所识别。 (3)转染各组质粒的HeLa细胞中， ELISA检测HeLa细胞在转染质粒48h后培养上清液中hscIL-23蛋白的含量，其中未转染组细胞培养上清为9.11±11.78pg/ml，转染pcDNA3.1(+)组的含量为10.47±9.04pg/ml，而转染pcDNA3.1(+)-pscIL-12组为254.34±2.77pg/ml，显著高于前两组（P0.01）。 (4)通过MTT法检测各组细胞培养上清刺激人淋巴细胞的增殖结果，未转染质粒组在570nm处的吸光度为0.553±0.038，PHA-P刺激组（阳性对照组）为1.968±0.018，转染pcDNA3.1(+)组为0.595±0.289，转染pcDNA3.1(+)-hscIL-23组组为1.135±0.048，显著高于未转染质粒组和转染pcDNA3.1(+)组（P0.01）。结论： (1)成功构建了原核表达质粒pET-28a(+)-hscIL-23和真核表达质粒pcDNA3.1(+)-hscIL-23； (2) pET-28a(+)-hscIL-23重组质粒在大肠杆菌内表达出目的蛋白； (3) pcDNA3.1(+)-hscIL-23在HeLa细胞中成功表达hscIL-23融合蛋白； (4)含真核表达hscIL-23蛋白的细胞培养上清能够刺激人淋巴细胞增殖。
[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.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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