本文選題：弓形蟲 + ROP18�。� 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的篩選神經(jīng)干細(xì)胞內(nèi)與弓形蟲ROP18相互作用的蛋白,為ROP18致病的分子機(jī)制研究奠定基礎(chǔ)。方法第一部分GST Pull-Down篩選C17.2神經(jīng)干細(xì)胞內(nèi)與弓形蟲ROP18的互作蛋白:(1)運(yùn)用RT-PCR擴(kuò)增ROP18基因;運(yùn)用Optigene TM密碼子優(yōu)化分析平臺對弓形蟲ROP18編碼基因進(jìn)行優(yōu)化,合成優(yōu)化后的全長基因(ROP18u);以優(yōu)化后的ROP18u基因?yàn)槟０?PCR擴(kuò)增去除N端信號肽和前功能區(qū)序列的截短ROP18片段(ROP18uc),將此3種基因分別克隆入pGEX-6P-1質(zhì)粒,構(gòu)建原核表達(dá)載體,所有重組質(zhì)粒均經(jīng)PCR、雙酶切和測序鑒定正確后,分別轉(zhuǎn)化大腸埃希菌BL21(DE3)誘導(dǎo)表達(dá),Western Blot鑒定重組蛋白,選取可溶性表達(dá)最高的菌種。(2)2%N2的DMEM/F12培養(yǎng)液誘導(dǎo)C17.2分化,收集細(xì)胞并提取總蛋白。(3)誘導(dǎo)表達(dá)ROP18u-GST蛋白和GST標(biāo)簽蛋白,利用體外Pull Down技術(shù)分別作用于分化C17.2總蛋白,通過對比獲得可能與ROP18u存在相互作用的蛋白,并進(jìn)行質(zhì)譜分析。第二部分免疫共沉淀技術(shù)篩選c17.2神經(jīng)干細(xì)胞內(nèi)與弓形蟲rop18的互作蛋白:(1)rt-pcr擴(kuò)增rop18基因,并無縫克隆至慢病毒載體pcdh-cmv-gfp中,經(jīng)pcr、酶切和測序鑒定正確后,與輔助質(zhì)粒pmlg/prre、prsv-rev和pmd2.g共轉(zhuǎn)染293t細(xì)胞包裝pcdh-cmv-gfp-rop18慢病毒,同時(shí)包裝pcdh-cmv-gfp空載體慢病毒作為對照組。(2)pcdh-cmv-gfp-rop18慢病毒和pcdh-cmv-gfp空載慢病毒分別感染c17.2,加以2%n2的dmem/f12誘導(dǎo)其分化,待綠色熒光大量表達(dá)時(shí)收集細(xì)胞并分別提取總蛋白。(3)合成兩段rop18抗原決定簇多肽,分別免疫新西蘭兔制備兩種兔抗rop18多克隆抗體,并進(jìn)行elisa和westernblot鑒定。(4)利用rop18多克隆抗體,分別同兩種慢病毒感染的分化c17.2總蛋白進(jìn)行免疫共沉淀,以期通過對比獲得在c17.2分化過程中可能與rop18相互作用的蛋白,與gstpull-down的結(jié)果相呼應(yīng)。結(jié)果第一部分:(1)經(jīng)誘導(dǎo)表達(dá)后對比發(fā)現(xiàn),優(yōu)化后的rop18u-gst可溶性蛋白表達(dá)量相較于rop18-gst明顯上升,經(jīng)優(yōu)化剪切后表達(dá)的rop18uc-gst蛋白可溶性表達(dá)量無顯著提升。(2)c17.2經(jīng)分化后,顯微鏡下可見胞體上出現(xiàn)明顯的形態(tài)、數(shù)量和長短各不相同的突起,呈樹枝分支狀。(3)gst標(biāo)簽蛋白pulldownc17.2分化總蛋白前后所得樣本經(jīng)sds-page檢測無明顯差異;rop18u-gst蛋白與c17.2總蛋白pulldown后的樣本相較于pulldown前,sds-page膠多出明顯蛋白條帶,經(jīng)質(zhì)譜分析獲得多個(gè)可能與rop18存在相互作用的鼠源性蛋白。第二部分:(1)重組慢病毒質(zhì)粒pcdh-cmv-gfp-rop18經(jīng)測序鑒定無誤,與輔助質(zhì)粒共轉(zhuǎn)染293t細(xì)胞,細(xì)胞出現(xiàn)明顯cpe且熒光顯微鏡下觀察到明顯的綠色熒光,包裝后第2天收獲并濃縮pcdh-cmv-gfp-rop18慢病毒;同樣方法包裝空載pCDH-CMV-GFP慢病毒。(2)兩種ROP18多克隆抗體,經(jīng)ELISA檢測,效價(jià)分別達(dá)到1:64000和1:16000;經(jīng)Western Blot鑒定,能夠特異性識別ROP18重組蛋白。(3)兩組慢病毒感染的C17.2均出現(xiàn)不同程度的CPE,熒光顯微鏡下發(fā)現(xiàn)明顯綠色熒光。(4)ROP18多克隆抗體未能拉下ROP18蛋白,經(jīng)Western Blot檢測,大部分ROP18蛋白仍殘留于免疫共沉淀后的上清中。結(jié)論(1)密碼子優(yōu)化未能顯著提高ROP18重組蛋白的表達(dá)量,但提高了可溶性蛋白的含量。(2)GST Pull-Down聯(lián)合質(zhì)譜結(jié)果表明ROP18與C17.2神經(jīng)干細(xì)胞內(nèi)的細(xì)胞骨架蛋白、組蛋白等多種蛋白間可能存在相互作用。(3)成功包裝ROP18慢病毒感染C17.2神經(jīng)干細(xì)胞,引起細(xì)胞病變,并穩(wěn)定表達(dá)ROP18。(4)成功制備兩種ROP18多克隆抗體,能夠特異性識別ROP18重組蛋白,但不適用于免疫共沉淀實(shí)驗(yàn)。
[Abstract]:Objective to screen the proteins interacting with Toxoplasma ROP18 in neural stem cells, and to lay the foundation for the molecular mechanism of ROP18. Method first part GST Pull-Down was used to screen the interaction proteins between C17.2 neural stem cells and Toxoplasma ROP18: (1) RT-PCR amplification of ROP18 gene and Optigene TM codon optimization analysis platform for Toxoplasma gondii The ROP18 encoding gene was optimized and the optimized full length gene (ROP18u) was synthesized. The optimized ROP18u base was amplified by PCR to remove the N terminal signal peptide and the truncated ROP18 fragment (ROP18uc) of the sequence of the anterior functional region. The 3 genes were cloned into pGEX-6P-1 plasmids, and the prokaryotic expression vector was constructed. All the recombinant plasmids were cut by PCR, double enzyme and the gene was constructed. After sequencing and identification, BL21 (DE3) induced expression of Escherichia coli was transformed, and Western Blot was used to identify the recombinant protein and select the highest soluble strain. (2) 2%N2 DMEM/F12 culture medium induced C17.2 differentiation, collected the cells and extracted the total protein. (3) the expression of ROP18u-GST protein and GST labelling protein was induced, and Pull Down technology in vitro was used respectively. The C17.2 protein was differentiated and the proteins interacting with ROP18u were obtained by contrast and mass spectrometric analysis. The interaction protein of C17.2 neural stem cells and Toxoplasma rop18 was screened by second part immunoprecipitation techniques: (1) RT-PCR amplification of rop18 gene, no slit cloning to the lentivirus carrier pcdh-cmv-gfp, PCR, enzyme digestion After the sequence identification was correct, the pcdh-cmv-gfp-rop18 lentivirus was packaged with the auxiliary plasmid pmlg/prre, prsv-rev and pmd2.g, and the pcdh-cmv-gfp no-load lentivirus was packaged as the control group. (2) pcdh-cmv-gfp-rop18 lentivirus and pcdh-cmv-gfp empty lentivirus infected C17.2 respectively, and 2%n2 dmem/f12 induced their differentiation to be green. When the color fluorescence was expressed in large quantities, the cells were collected and the total protein was extracted respectively. (3) two segments of rop18 antigen determinant polypeptide were synthesized and two Rabbit anti rop18 polyclonal antibodies were prepared from New Zealand rabbits, respectively, and ELISA and Westernblot were identified. (4) rop18 polyclonal antibody was used to immunize the total C17.2 total protein of two kinds of lentivirus infection respectively. Precipitation, in order to obtain the protein that may interact with rop18 during the differentiation of C17.2, echoes with the results of gstpull-down. Results the first part: (1) after the induction of expression, the optimized expression of rop18u-gst soluble protein was significantly higher than that of rop18-gst, and the rop18uc-gst protein expressed after the optimized shear was expressed. There was no significant increase in soluble expression. (2) after the differentiation of C17.2, the apparent morphology was found on the cell body under the microscope, and the number and length were different. (3) there was no significant difference between the samples before and after the GST labeling protein pulldownc17.2 differentiation total protein; rop18u-gst protein and C17.2 total protein pulldown After the samples were compared with the pulldown, the SDS-PAGE glue had more obvious protein bands, and several mouse derived proteins that might interact with rop18 were obtained by mass spectrometry. The second part: (1) the recombinant lentivirus plasmid pcdh-cmv-gfp-rop18 was sequenced and identified, and the 293T cells were co transfected with the auxiliary plasmids, and the cells showed a clear CPE and fluorescence microscope. The obvious green fluorescence was observed, and pcdh-cmv-gfp-rop18 lentivirus was harvested and concentrated at second days after packaging; the same method was used to package the empty pCDH-CMV-GFP lentivirus. (2) two ROP18 polyclonal antibodies were detected by ELISA, and the titers were 1:64000 and 1:16000 respectively. The ROP18 recombinant protein could be identified by Western Blot. (3) two groups of lentiviruses. The infected C17.2 showed different degrees of CPE and found obvious green fluorescence under the fluorescence microscope. (4) ROP18 polyclonal antibody failed to pull down ROP18 protein, and most of ROP18 protein remained in the supernatant after immunoprecipitation after Western Blot. Conclusion (1) optimization of codon did not significantly increase the expression of ROP18 recombinant protein, but improved The content of soluble protein. (2) GST Pull-Down combined mass spectrometry results showed that there might be interaction between the cytoskeleton protein, histone and other proteins in ROP18 and C17.2 neural stem cells. (3) the successful packaging of ROP18 lentivirus infected C17.2 neural stem cells, causing cytopathic disease, and stabilizing the expression of ROP18. (4) to produce two kinds of ROP18. The cloned antibody can specifically recognize ROP18 recombinant protein, but it is not suitable for coprecipitation.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R382.5

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1 陳冠軍,邢金良,商澎,陳志南;外源基因在大腸桿菌中表達(dá)效率的影響因素[J];世界最新醫(yī)學(xué)信息文摘;2004年03期

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