【摘要】： 目的:構(gòu)建豬帶絳蟲(chóng)六鉤蚴期密碼子優(yōu)化TSOL18基因的重組核酸疫苗優(yōu)化PVAX1/TSOL18,觀察其在體外CHO-K1倉(cāng)鼠卵巢細(xì)胞亞株中的表達(dá)以及經(jīng)肌肉接種后在小鼠骨骼肌內(nèi)的表達(dá),為豬帶絳蟲(chóng)囊尾蚴病DNA疫苗的進(jìn)一步研究奠定基礎(chǔ)。方法:按照哺乳動(dòng)物的密碼子使用偏好,對(duì)豬帶絳蟲(chóng)六鉤蚴TSOL18基因進(jìn)行優(yōu)化,使用GeneOptimizer軟件對(duì)TSOL18基因的氨基酸編碼基因進(jìn)行優(yōu)化,但不改變氨基酸。根據(jù)優(yōu)化后的基因序列,由南京金思特科技有限公司進(jìn)行全基因合成。利用PCR擴(kuò)增該基因序列,將其插入真核表達(dá)載體PVAX1,經(jīng)酶切,測(cè)序鑒定。大量提取重組質(zhì)粒,將重組質(zhì)粒優(yōu)化PVAX1/TSOL18經(jīng)梭華-SofastTM介導(dǎo),體外轉(zhuǎn)染CHO-K1細(xì)胞,通過(guò)RT-PCR、細(xì)胞裂解物的SDS-PAGE電泳及Western-blotting分析檢測(cè)其在細(xì)胞內(nèi)是否有表達(dá),再將密碼子優(yōu)化的PVAX1/TSOL18和PVAX1/TSOL18進(jìn)行體內(nèi)轉(zhuǎn)染繼以免疫組化法觀察其在肌肉組織內(nèi)的表達(dá)效果并作比較。結(jié)果:優(yōu)化密碼子的PVAX1/TSOL18重組質(zhì)粒經(jīng)酶切電泳和測(cè)序結(jié)果與預(yù)期設(shè)計(jì)的基因序列一致。真核細(xì)胞轉(zhuǎn)染后通過(guò)RT-PCR結(jié)果顯示目的條帶約為414bp;真核細(xì)胞轉(zhuǎn)染后通過(guò)SDS-PAGE電泳及Western-blotting檢測(cè)表明能夠在CHO-K1細(xì)胞中表達(dá)出約為18kDa的目的蛋白。免疫組化染色后在重組質(zhì)粒注射部位肌肉組織的肌纖維內(nèi)顯示棕黃色陽(yáng)性分泌顆粒,并且優(yōu)化PVAX1/TSOL18組比PVAX1/TSOL18組棕黃色陽(yáng)性分泌顆粒多,空載體組與空白對(duì)照組骨骼肌組織未見(jiàn)明顯的棕黃色顆粒。結(jié)論:成功構(gòu)建PVAX1/TSOL18密碼子優(yōu)化基因的真核表達(dá)質(zhì)粒;并且初步揭示了優(yōu)化PVAX1/TSOL18在體內(nèi)和體外的表達(dá),優(yōu)化PVAX1/TSOL18比PVAX1/TSOL18在體內(nèi)表達(dá)量更多,為進(jìn)一步在體驗(yàn)證密碼子優(yōu)化PVAX1/TSOL18保護(hù)性和進(jìn)行大規(guī)�，F(xiàn)場(chǎng)研究奠定了基礎(chǔ)。
[Abstract]:Objective: to construct a recombinant nucleic acid vaccine to optimize the TSOL18 gene of Taenia solium and to observe its expression in CHO-K1 hamster ovarian cell subline in vitro and in the skeletal muscle of mice after intramuscular inoculation. To lay a foundation for further research on DNA vaccine of cysticercosis solium. Methods: according to the preference of mammalian codon, the TSOL18 gene of Taenia solium was optimized, and the amino acid encoding gene of TSOL18 gene was optimized by GeneOptimizer software, but the amino acid was not changed. According to the optimized gene sequence, the whole gene synthesis was carried out by Nanjing Kinster Technology Co., Ltd. The gene was amplified by PCR and inserted into eukaryotic expression vector PVAX1,. A large number of recombinant plasmids were extracted, and the optimized PVAX1/TSOL18 was transfected into CHO-K1 cells by Sova-SofastTM in vitro. The expression of RT-PCR, cell lysates was detected by SDS-PAGE electrophoresis and Western-blotting analysis. Then the codon optimized PVAX1/TSOL18 and PVAX1/TSOL18 were transfected in vivo and then the expression of codon optimized PVAX1/TSOL18 and PVAX1/TSOL18 in muscle tissue was observed by immunohistochemical method and compared. Results: the PVAX1/TSOL18 recombinant plasmid with optimized codon was sequenced by restriction endonuclease electrophoresis. The result of RT-PCR showed that the target band of eukaryotic cells was about 414bp.After the transfection of eukaryotic cells, SDS-PAGE electrophoresis and Western-blotting detection showed that the target protein was about 18kDa in CHO-K1 cells. After immunohistochemical staining, brown positive secretory granules were found in muscle fibers of muscle tissue at the injection site of recombinant plasmid, and there were more brown positive secretory granules in optimized PVAX1/TSOL18 group than in PVAX1/TSOL18 group. There were no obvious brown granules in skeletal muscle of empty carrier group and blank control group. Conclusion: the eukaryotic expression plasmid of PVAX1/TSOL18 codon optimization gene was successfully constructed. It was revealed that the expression of PVAX1/TSOL18 in vivo and in vitro was optimized, and the expression of PVAX1/TSOL18 was more than that of PVAX1/TSOL18 in vivo, which laid a foundation for further codon optimization of PVAX1/TSOL18 protection and large-scale field research.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 劉立營(yíng);基于全基因合成技術(shù)的南極假絲酵母脂肪酶B基因的密碼子優(yōu)化研究[D];華中科技大學(xué);2011年

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本文編號(hào)：2310450