Liang Nannan, Zhang Lijun, Zhao Haiquan, Liu Zhongjian, Luo Huanliang, Lin Yanxing, Liu Xiaoxiao
College of Life Science, Anhui Agricultural University, Hefei 230036, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Jun;29(6):625-8.
To construct the prokaryotic expression vector of phytoplasma immunodominant membrane protein A (IdpA) in prokaryotic cell, express and purify the IdpA and prepare its antiserum.
With the recombinant plasmid pMD18-T-IdpA as templates, IdpA gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a(+) by endonuclease reaction and T4 DNA ligase reaction. Then the recombinant plasmid pET-28a(+)-IdpA was transformed into E.coli BL21 (DE3). After confirmed by PCR and double enzyme digestion, the recombinant protein IdpA was expressed under IPTG induction and purified. The purified product was used to immunize BALB/c mice to prepare its antiserum. IdpA-specific mouse antiserum was identified by ELISA and Westerrn blotting.
The prokaryotic vectors of pET-28a(+)-IdpA were constructed successfully and the recombinant protein IdpA was induced to express stably in the E.coli BL21. The purity of IdpA was up to over 90%. In the BALB/c mice immunized by the purified IdpA, the titre of IdpA-specific antiserum was as high as 1:320 000.
The recombinant protein IdpA was expressed successfully in E.coli and the IdpA-specific antiserum was prepared.
构建植原体免疫显性膜蛋白A（IdpA）的原核表达载体，在原核细胞中表达并纯化IdpA，制备其抗血清。
以重组质粒pMD18-T-IdpA为模板，通过PCR扩增IdpA基因，并经内切酶反应和T4 DNA连接酶反应克隆至原核表达载体pET-28a(+)中。然后将重组质粒pET-28a(+)-IdpA转化至大肠杆菌BL21(DE3)。经PCR和双酶切鉴定后，重组蛋白IdpA在IPTG诱导下表达并纯化。纯化产物用于免疫BALB/c小鼠以制备其抗血清。通过ELISA和Western印迹鉴定IdpA特异性小鼠抗血清。
成功构建了pET-28a(+)-IdpA原核表达载体，重组蛋白IdpA在大肠杆菌BL21中稳定诱导表达。IdpA纯度高达90%以上。在经纯化的IdpA免疫的BALB/c小鼠中，IdpA特异性抗血清效价高达1:320 000。
重组蛋白IdpA在大肠杆菌中成功表达，并制备了IdpA特异性抗血清。
