裂谷熱（Rift Valley fever，RVF）是由裂谷熱病毒（Rift Valley fever virus，RVFV）引起的一種烈性人獸共患傳染病。RVFV囊膜蛋白Gn可誘導(dǎo)產(chǎn)生中和抗體，是RVFV檢測方法和疫苗研究的重要抗原靶標(biāo)。本研究通過分析蛋白抗原位點(diǎn)信息，構(gòu)建包含Gn蛋白兩個(gè)主要抗原區(qū)域的重組表達(dá)載體，隨后將質(zhì)粒轉(zhuǎn)化至BL21感受態(tài)細(xì)胞，以IPTG誘導(dǎo)重組蛋白表達(dá)并優(yōu)化蛋白表達(dá)條件，通過Western Blot鑒定重組蛋白；將重組蛋白免疫BALB/c小鼠，制備多克隆抗體，并以ELISA、Western Blot、IFA檢測多克隆抗體的反應(yīng)性。結(jié)果顯示：誘導(dǎo)表達(dá)的Gn重組蛋白分子質(zhì)量約為45 kDa；蛋白表達(dá)條件優(yōu)化為IPTG終濃度0.25 mmol/L，誘導(dǎo)時(shí)間5 h；Western Blot鑒定發(fā)現(xiàn)蛋白成功表達(dá)。通過ELISA測定小鼠三免后血清抗體，結(jié)果發(fā)現(xiàn)抗體效價(jià)大于1:51 200；Western Blot檢測顯示，制備的多抗血清能與重組蛋白發(fā)生反應(yīng)；進(jìn)一步的IFA檢測結(jié)果表明，制備的多克隆抗體可與真核質(zhì)粒轉(zhuǎn)染細(xì)胞中表達(dá)的Gn蛋白反應(yīng)。本研究獲得的Gn重組蛋白及制備的多克隆抗體為后續(xù)RVFV檢測方法的建立奠定了基礎(chǔ)。
Tandem Expression of the Major Antigenic Areas of RVFV Gn Protein and the Preparation of Polyclonal Antibodies
Rift Valley fever（RVF）is a virulent zoonotic disease caused by Rift Valley fever virus（RVFV）. The envelope protein Gn of RVFV is an important antigen target for the detection method development and vaccine research，as it could induce the production of neutralizing antibodies. In the study，the recombinant expression vector containing two major antigen areas of Gn protein was constructed through analyzing the information of protein antigen sites，then the plasmid was transformed into BL21 cells，the recombinant protein expression was induced by IPTG，followed by optimization of relevant conditions，the recombinant protein was identified by Western Blot and then vaccinated into BALB/c mice to prepare the polyclonal antibodies that were tested by ELISA，Western Blot and IFA. The results showed that the molecular weight of the induced Gn recombinant protein was about 45 kDa；the expression conditions were optimized as follows：the final concentration of IPTG was 0.25 mmol/L with the induction time of 5 h；and the protein was successfully expressed as identified by Western Blot. The antibody titer in vaccinated mice was higher than 1:51 200 as tested by ELISA；it was shown by Western Blot that，the prepared polyclonal antiserum could react with the recombinant protein；and the polyclonal antibodies could react with the Gn protein expressed in eukaryotic transfection cells as detected by IFA. In conclusion，future development of RVF detection methods was provided with a basis by the expression of Gn protein and preparation of polyclonal antibodies.
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