摘要
目的 原核表达、纯化腺相关病毒9(adeno-associated virus 9,AAV9)衣壳可变区蛋白,并制备针对AAV9衣壳可变区的兔多克隆抗体。方法 设计并合成编码AAV9衣壳蛋白可变区的DNA序列,插入原核表达载体pET-30a获得质粒pET-30a-AAV-VR,转化E.coli BL21(DE3),IPTG诱导表达多价抗原肽,在变性条件下经Ni-NTA树脂纯化,透析复性后将AAV9可变区蛋白免疫雄性日本大耳白兔,制备多克隆抗体,间接ELISA法检测抗体效价,Western blot及细胞免疫荧光法检测抗体的特异性。结果 表达AAV9衣壳可变区蛋白的重组原核表达质粒pET-30a-AAV-VR经XhoⅠ和BglⅡ双酶切鉴定构建正确。表达的蛋白相对分子质量约为20 000,纯化后可被His标签抗体识别。制备的兔多克隆抗体效价为1∶10 240 000,能特异性识别AAV9衣壳蛋白。结论 成功表达了AAV9衣壳可变区蛋白,并制备了高效价的兔多克隆抗体,为后续AAV载体开发以及AAV生物学功能研究奠定了基础。
Objective To express and purify the protein of variable region of adeno-associated virus 9(AAV9) capsid in prokaryotic cells,and prepare rabbit polyclonal antibody against it.Methods DNA sequence encoding variable region of AAV9 capsid protein was designed,synthesized and inserted into prokaryotic expression vector pET-30a.The obtained plasmid pET-30a-AAV-VR was transformed to E.coli BL21(DE3),induced by IPTG to express the multivalent antigenic peptide and purified by Ni-NTA resin under denaturation conditions.Male Japanese large-eared white rabbits were immunized with the AAV9 variable region protein after dialysis and renaturation to prepare polyclonal antibody,which was determined for the antibody potency by indirect ELISA,and for the specificity by Western blot and cellular immunofluorescence.Results The recombinant prokaryotic expression plasmid pET-30a-AAV-VR expressing the variable region of AAV9 capsid was constructed correctly as identified by XhoⅠ and BglⅡ digestion.The expressed protein was recognized by His tag antibody after purification with a relative molecular mass of about 20 000.The potency of rabbit polyclonal antibody was 1∶10 240 000,which specifically recognize AAV9 capsid protein.Conclusion The capsid variable region protein of AAV9 was successfully expressed and rabbit polyclonal antibody with high potency was prepared,which laid a foundation of the subsequent development of AAV vector and the research of AAV biological function.
作者
李舒月
张浩
张雄洲
赵云汉
杨建林
曹春雨
吕亚丰
LI Shuyue;ZHANG Hao;ZHANG Xiongzhou;ZHAO Yunhan;YANG Jianlin;CAO Chunyu;LV Yafeng(Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,College of Basic Medical Sciences,China Three Gorges University,Yichang 443002,Hubei Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2024年第7期837-842,共6页
Chinese Journal of Biologicals
基金
湖北省教育厅中青年人才项目(Q20211207)
湖北省老年胃肠癌精准防治临床医学研究中心开放基金(2022EGC-05)。
关键词
腺相关病毒9
衣壳可变区
原核表达
多克隆抗体
Adeno-associated virus 9(AAV9)
Capsid variable region
Prokaryotic expression
Polyclonal antibody
