腺相关病毒衣壳保守区重组蛋白的原核表达及其多克隆抗体的制备
[Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody].
作者信息
Li S, Cao C, Zhang H, Li Y, Zhang X, Yang Z, Xia Y, Wang L, Lü Y
机构信息
Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University Medical College, Yichang 443000, China.
Hubei Provincial Key Laboratory of Occurrence and Intervention of Rheumatic Diseases, Affiliated Hospital of Hubei University for Nationalities, Enshi 445000, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Jun 20;42(6):944-948. doi: 10.12122/j.issn.1673-4254.2022.06.20.
OBJECTIVE
To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody.
METHODS
The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay.
RESULTS
The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10.
CONCLUSION
We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
目的
在原核细胞中表达和纯化腺相关病毒（AAV）衣壳保守区的抗原肽，并制备其兔多克隆抗体。
方法
合成编码AAV衣壳蛋白保守区的DNA序列，并克隆到载体pET30a中，获得用于原核表达和纯化保守肽的质粒pET30a-AAV-CR。采用考马斯亮蓝染色和蛋白质印迹法鉴定AAV保守肽。用AAV保守区蛋白免疫日本大耳白兔制备多克隆抗体，以注射PBS的兔子作为对照组。用酶联免疫吸附测定法测定抗体效价，并用蛋白质印迹法和免疫荧光测定法评估抗体识别AAV1-AAV10衣壳蛋白序列的性能。
结果
成功构建了质粒pET30a-AAV-CR，获得了相对分子质量为17000的重组蛋白。纯化后的蛋白诱导兔产生了针对AAV衣壳保守区的抗体，纯化抗体的效价达到1:320 000。这些抗体能够识别AAV1-AAV10的多种衣壳蛋白序列。
结论
我们成功从兔体内获得了抗AAV衣壳保守区蛋白的多克隆抗体，这有助于未来AAV载体开发和AAV生物学功能的研究。
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