摘要
目的:制备嵌合结肠癌新抗原Adpgk的乙肝病毒核心抗原(HBcAg)病毒样颗粒(VLP)。方法:通过重叠PCR将新抗原编码序列插入截短型HBcAg(1~149 aa)第78、79位氨基酸编码序列之间,并将融合基因克隆至原核表达载体pET-22b(+),转化大肠杆菌BL21(DE3),经IPTG诱导表达后,利用亲和层析与超滤管纯化浓缩目的蛋白,通过Western印迹、粒径分析与透射电镜成像鉴定嵌合病毒样颗粒。结果:构建了pET-22b(+)重组表达载体并转入大肠杆菌BL21(DE3),SDS-PAGE结果显示在30℃经1 mmol/L IPTG诱导5 h即可获得高浓度的可溶性蛋白,通过亲和层析与超滤浓缩得到了纯度良好的目的蛋白;Western印迹结果表明连有His标签的目的蛋白获得表达且浓度较高;透射电镜成像可见密集的大小均一、空心的球形病毒样颗粒结构。结论:制备出浓度与纯度良好且组装正确的嵌合HBcAg VLP。
Objective:To prepare the chimeric virus-like particles(VLPs)of hepatitis B virus core antigen(HBcAg)presenting colon cancer neoantigen Adpgk.Methods:The coding sequence of neoantigen was inserted into position between amino acids 78 and 79 of truncated HBcAg 1~149 aa by overlap PCR,then the fusion gene was cloned into pET-22b(+)vector and the recombinant vector was subsequently transformed into E.coli BL21(DE3)and induced by IPTG.Expressed fusion protein was purified and concentrated by affinity chromatography and ultrafiltration,respectively.The formation of chimeric virus-like particles was identified by Western blot,particle size analysis,and transmission electron microscopy.Results:The recombinant expression vector pET-22b(+)was successfully constructed and transformed into E.coli BL21(DE3).The soluble target protein was highly expressed after induction with 1 mmol/L IPTG for 5 h at 30℃.The target protein with good purity was obtained by affinity chromatography and ultrafiltration.The His-tagged target fusion protein was successfully expressed at a higher concentration as confirmed by Western blot.Dense spherical VLPs with uniform size and hollow structure could be seen under transmission electron microscope.Conclusion:Correctly assembled chimeric HBcAg VLPs with good concentration and purity were successfully prepared.
作者
赵振伯
杨怡姝
马洪涛
盛望
ZHAO Zhen-Bo;YANG Yi-Shu;MA Hong-Tao;SHENG Wang(College of Life Science and Bioengineering,Beijing University of Technology,Beijing 100124,China)
出处
《生物技术通讯》
CAS
2020年第1期1-6,共6页
Letters in Biotechnology
基金
国家自然科学基金(31770999)。
关键词
结肠癌
新抗原
乙肝病毒核心抗原
原核表达
colon cancer
neoantigen
hepatitis B virus core antigen(HBcAg)
prokaryotic expression
