【摘要】：為了研發(fā)植物來源的輪狀病毒疫苗,采用模式植物擬南芥作為生物反應(yīng)器,將輪狀病毒主要中和抗原基因VP7融合CTB,通過農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化野生型擬南芥,獲得了含有VP7-CTB的轉(zhuǎn)基因擬南芥穩(wěn)定株系,并分析其種子表達(dá)蛋白的免疫原性。首先,根據(jù)擬南芥密碼子的偏好性,設(shè)計(jì)合成優(yōu)化的VP7和CTB基因,構(gòu)建植物表達(dá)載體pPHAP1301-his-VP7-linker-CTB;再通過農(nóng)桿菌介導(dǎo)的Floral Dip法轉(zhuǎn)化擬南芥,收獲T0代種子后播種,噴用草銨膦篩選轉(zhuǎn)基因擬南芥獲得T1代種子,重復(fù)上述過程最終獲得T3代擬南芥種子,通過RT-PCR、W estern-bolt、ELISA等進(jìn)行分析。結(jié)果表明,his-VP7-linker-CTB基因已成功整合到擬南芥的基因組并進(jìn)行了轉(zhuǎn)錄和蛋白表達(dá),并且VP7蛋白占總可溶性蛋白的0.39%,即轉(zhuǎn)基因擬南芥種子中所含目的蛋白為52.65 g/g。最后,用轉(zhuǎn)基因擬南芥穩(wěn)定株系種子中的可溶性蛋白口服免疫小鼠,使小鼠獲得免疫原性;乳鼠攻毒試驗(yàn)證明,母鼠免疫后產(chǎn)生的抗體能夠有效傳遞給子代,具有很好的保護(hù)作用。
[Abstract]:In order to develop a plant-derived rotavirus vaccine, Arabidopsis thaliana, a model plant, was used as a bioreactor to fuse the main neutralizing antigen gene VP7 of rotavirus into wild type Arabidopsis thaliana by Agrobacterium tumefaciens mediated method. A transgenic Arabidopsis thaliana stable strain containing VP7-CTB was obtained and the immunogenicity of its seed expression protein was analyzed. Firstly, according to the preference of Arabidopsis codon, the optimized VP7 and CTB genes were designed and synthesized, and the plant expression vector pPHAP1301-his-VP7-linker-CTB; was constructed. Then Arabidopsis thaliana was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated Floral Dip method, then seeded after harvesting T0 generation seeds, then T1 generation seeds were obtained by spraying ammonium phosphate to screen transgenic Arabidopsis thaliana seeds, and T3 generation Arabidopsis thaliana seeds were obtained by repeating the above process. Through RT-PCR,W estern-bolt, ELISA and so on carry on the analysis. The results showed that his-VP7-linker-CTB gene had been successfully integrated into the genome of Arabidopsis thaliana and transcribed and expressed, and VP7 protein accounted for 0.39% of the total soluble protein. That is to say, the target protein contained in transgenic Arabidopsis thaliana seeds was 52.65 g 路kg ~ (- 1). Finally, the mice were immunized with soluble proteins from the seeds of transgenic Arabidopsis thaliana stable strain to obtain immunogenicity. The challenge test of neonatal mice showed that the antibodies produced by immunization of female mice could be effectively transmitted to their offspring and had a good protective effect.
【作者單位】： 吉林農(nóng)業(yè)大學(xué)教育部生物反應(yīng)器與藥物開發(fā)工程研究中心;吉林農(nóng)業(yè)科技學(xué)院中藥學(xué)院;吉林農(nóng)業(yè)大學(xué)中藥材學(xué)院;溫州大學(xué);
【基金】：國(guó)家高技術(shù)研究發(fā)展計(jì)劃(863)項(xiàng)目(2011AA100606)
【分類號(hào)】：Q943.2;S859.797
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本文編號(hào)：2483888