本文選題：重癥肌無力 + 肌肉特異性酪氨酸激酶　； 參考：《延邊大學(xué)》2014年碩士論文

【摘要】：目的：將構(gòu)建好的肌肉特異性酪氨酸激酶(MuSK)-紅色熒光蛋白(mCherry)融合蛋白(MuSK-mCherry)基因,轉(zhuǎn)染在人胚腎HEK293細(xì)胞中進(jìn)行表達(dá),為后續(xù)檢測重癥肌無力患者(MG) MuSK抗體制備抗原。 方法：將轉(zhuǎn)化有pMT/BiP/V5-His (MuSK-mCherry)的E coli DH5a擴(kuò)大培養(yǎng),提取出質(zhì)粒,電泳后置于凝膠成像系統(tǒng)中觀察其位置確認(rèn)是否含有pMT/BiP/V5-His (MuSK-mCherry)質(zhì)粒。分別在放有爬片的6孔細(xì)胞培養(yǎng)板和普通6孔細(xì)胞培養(yǎng)板中培養(yǎng)人胚腎HEK392細(xì)胞,將提取出的質(zhì)粒轉(zhuǎn)染至人胚腎HEK293細(xì)胞中。轉(zhuǎn)染后取放有爬片的6孔細(xì)胞培養(yǎng)板第24小時(shí)的細(xì)胞,通過抗熒光猝滅封片劑封片,凝固后應(yīng)用共聚焦顯微鏡在激發(fā)波長為587nm、發(fā)射波長為610nm處觀察熒光圖像確認(rèn)轉(zhuǎn)染表達(dá)的情況。在轉(zhuǎn)染后48小時(shí)內(nèi)每6個(gè)小時(shí)為一時(shí)間將轉(zhuǎn)染后的普通6孔細(xì)胞培養(yǎng)板中的細(xì)胞提取凍存為細(xì)胞團(tuán)。將細(xì)胞團(tuán)統(tǒng)一進(jìn)行細(xì)胞裂解提取出蛋白后,應(yīng)用免疫印跡檢測技術(shù)檢測表達(dá)的融合蛋白,觀察條帶確認(rèn)細(xì)胞表達(dá)情況及轉(zhuǎn)染后每個(gè)時(shí)間段的表達(dá)差異。 結(jié)果：通過電泳檢測于E coli DH5a中提取出的質(zhì)粒溶液在凝膠成像系統(tǒng)中觀察,發(fā)現(xiàn)5647bp大小條帶,與理論條帶位置符合。在HE293細(xì)胞中轉(zhuǎn)染后通過免疫熒光圖像觀察到有紅色熒光蛋白,于每6小時(shí)的細(xì)胞團(tuán)中提取的蛋白進(jìn)行免疫印跡試驗(yàn),可觀察到72.4KDa大小條帶,與理論條帶位置符合,并觀察到轉(zhuǎn)染后每個(gè)時(shí)間段的表達(dá)有差異。 結(jié)論：含有MuSK-mCherry融合蛋白的載體質(zhì)粒pMT/BiP/V5-His (MuSK-mCherry)可在HEK293細(xì)胞中進(jìn)行表達(dá),獲得了MuSK-mCherry融合蛋白。這為后續(xù)檢查MG患者M(jìn)uSKAb準(zhǔn)備了抗原。
[Abstract]:Aim: to transfect the muscle-specific tyrosine kinase mCherryprotein muSK-mCherry) gene into human embryonic kidney HEK293 cells, and to prepare the antigen for the subsequent detection of MuSK antibody in patients with myasthenia gravis. Methods: the E. coli DH5a transformed with pMT/BiP/V5-His mSK-mCherrywas expanded and the plasmids were extracted, and then the plasmids were extracted by gel imaging system. The position of the plasmids was detected by gel imaging system to confirm the existence of pMT/BiP/V5-His MuSK-mCherryplasmids. Human embryonic kidney HEK392 cells were cultured in 6-well cell culture plate and 6-well cell culture plate respectively. The extracted plasmids were transfected into human embryonic kidney HEK293 cells. After transfection, the cells of the 6-well cell culture plate with crawling tablets were removed for 24 hours, and the cells were sealed with anti-fluorescence quenching tablets. After solidification the transfection expression was confirmed by confocal microscope at the excitation wavelength of 587 nm and the emission wavelength of 610nm. After 48 hours of transfection, the cells from the normal 6-well cell culture plate were extracted and frozen into a cell mass every 6 hours after transfection. The fusion protein was detected by Western blotting technique after the cell mass was extracted by cell lysis. The expression of the fusion protein was confirmed by bands and the difference of expression in each period after transfection was observed. Results: the plasmids extracted from E coli DH5a were observed in gel imaging system by electrophoresis. The size bands of 5647bp were found to be consistent with the theoretical bands. After transfection in HE293 cells, red fluorescent protein was observed by immunofluorescence image, and the protein extracted from the cell mass every 6 hours was detected by Western blotting test. The size bands of 72.4KDa were observed, which coincided with the position of the theoretical bands. It was observed that there were differences in expression at each time after transfection. Conclusion: the vector plasmid pMT/BiP/V5-His MuSK-mCherrycontaining MuSK-mCherry fusion protein can be expressed in HEK293 cells and the MuSK-mCherry fusion protein is obtained. This prepares antigens for follow-up examination of MuSKAb in MG patients.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R746.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 劉文雯;李勇;廖思明;周興;朱浩君;黃日波;;肌肉特異性激酶基因的克隆、表達(dá)和純化[J];生物技術(shù);2012年03期

2 馬曉偉;楊麗;楊春生;張大啟;翟，

本文編號：1927602