生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 157-163.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1076
汪巧菊(), 胡雨萌, 温亚亚, 宋丽, 孟闯, 潘志明(), 焦新安
收稿日期:
2021-08-21
出版日期:
2022-03-26
发布日期:
2022-04-06
作者简介:
汪巧菊,女,硕士研究生,研究方向:遗传学;E-mail: 基金资助:
WANG Qiao-ju(), HU Yu-meng, WEN Ya-ya, SONG Li, MENG Chuang, PAN Zhi-ming(), JIAO Xin-an
Received:
2021-08-21
Published:
2022-03-26
Online:
2022-04-06
摘要:
旨在获得具有免疫活性的SARS-CoV-2 S1蛋白,为COVID-19的免疫预防及临床诊断提供较好的候选抗原。该研究利用PCR技术扩增出刺突蛋白(Spike,S)的S1亚基序列,通过同源重组法将目的片段克隆至冷休克表达载体pCold I,转化至大肠杆菌BL21感受态细胞内诱导表达S1蛋白,经Ni2+柱亲和层析纯化后,通过SDS-PAGE和Western blotting鉴定目的蛋白的表达情况。结果表明,已成功表达含His标签的S1蛋白,并且已获得浓度和纯度较高、能与S1多克隆抗体反应的目的蛋白。此外,蛋白刺激细胞的结果表明,原核表达的S1蛋白免疫原性良好,能诱导小鼠巨噬细胞上调表达多种细胞因子和趋化因子,促进RAW264.7细胞产生免疫反应。综上,该研究成功构建了pCold I-S1重组质粒,获得了SARS-CoV-2的良好候选抗原S1蛋白。
汪巧菊, 胡雨萌, 温亚亚, 宋丽, 孟闯, 潘志明, 焦新安. 新型冠状病毒S1蛋白的表达及活性鉴定[J]. 生物技术通报, 2022, 38(3): 157-163.
WANG Qiao-ju, HU Yu-meng, WEN Ya-ya, SONG Li, MENG Chuang, PAN Zhi-ming, JIAO Xin-an. Expression and Activity Identification of SARS-CoV-2 S1 Protein[J]. Biotechnology Bulletin, 2022, 38(3): 157-163.
基因 Gene | 引物序列 Primer sequence(5'-3') | 产物大小 Product size/bp |
---|---|---|
IL-1β | F:GCCCATCCTCTGTGACTCAT | 230 |
R:AGGCCACAGGTATTTTGTCG | ||
TNF-α | F:AGCCCCCAGTCTGTATCCTT | 212 |
R:CTCCCTTTGCAGAACTCAGG | ||
MIP-2 | F:AAGTTTGCCTTGACCCTGAA | 180 |
R:AGGCACATCAGGTACGATCC | ||
IP-10 | F:GGATGGCTGTCCTAGCTCTG | 211 |
R:ATAACCCCTTGGGAAGATGG | ||
β-actin | F:AGCCATGTACGTAGCCATCC | 228 |
R:CTCTCAGCTGTGGTGGTGAA |
表1 引物序列
Table 1 Primer sequence
基因 Gene | 引物序列 Primer sequence(5'-3') | 产物大小 Product size/bp |
---|---|---|
IL-1β | F:GCCCATCCTCTGTGACTCAT | 230 |
R:AGGCCACAGGTATTTTGTCG | ||
TNF-α | F:AGCCCCCAGTCTGTATCCTT | 212 |
R:CTCCCTTTGCAGAACTCAGG | ||
MIP-2 | F:AAGTTTGCCTTGACCCTGAA | 180 |
R:AGGCACATCAGGTACGATCC | ||
IP-10 | F:GGATGGCTGTCCTAGCTCTG | 211 |
R:ATAACCCCTTGGGAAGATGG | ||
β-actin | F:AGCCATGTACGTAGCCATCC | 228 |
R:CTCTCAGCTGTGGTGGTGAA |
图1 S1基因PCR产物电泳分析 M:DL5000 DNA marker;1-2:S1基因PCR扩增产物
Fig. 1 Electrophoresis analysis of S1 gene PCR product M:DL5000 DNA marker. 1-2:PCR product of S1 gene
图2 重组质粒pCold I-S1的PCR鉴定 M:DL5000 DNA marker;1-2:pCold I-S1质粒的PCR扩增产物
Fig. 2 PCR identification of recombinant plasmid pCold I-S1 M:DL5000 DNA marker;1-2:PCR product of pCold I-S1
图3 S1蛋白的SDS-PAGE分析 M:Protein marker;1:BL21(pColdⅠ)诱导菌;2:BL21(pColdⅠ-S1)未诱导菌;3:BL21(pColdⅠ-S1)诱导上清;4:BL21(pColdⅠ-S1)诱导沉淀
Fig. 3 SDS-PAGE analysis of S1 protein M:Protein marker. 1:Induced BL21(pColdⅠ). 2:Non-induced BL21(pColdⅠ-S1). 3:Supernatant of induced BL21(pColdⅠ-S1). 4:Precipi-tation of induced BL21(pColdⅠ-S1)
图4 纯化后S1蛋白的SDS-PAGE分析 M:Protein marker;1:BL21(pCold Ⅰ)诱导菌;2:BL21(pColdⅠ-S1)诱导上清;3:BL21(pColdⅠ-S1)诱导沉淀;4-5:纯化后S1蛋白
Fig. 4 SDS-PAGE analysis of purified S1 protein M:Protein marker. 1:Induced BL21(pColdⅠ). 2:Supernatant of induced BL21 (pColdⅠ-S1). 3:Precipitation of induced BL21(pColdⅠ-S1). 4-5:The purified S1 protein
图5 纯化后S1蛋白的Western blotting鉴定 M:Protein marker;1:BL21(pColdⅠ)诱导菌;2:纯化后S1蛋白
Fig. 5 Western blotting identification of purified S1 protein M:Protein marker. 1:Induced BL21(pColdⅠ). 2:The purified S1 protein
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