新型基因組編輯蛋白FnCpf1的表達(dá)及其功能的初步探討
發(fā)布時間：2019-03-22 19:09
【摘要】：目的構(gòu)建土拉弗朗西絲菌novicida亞種Cpf1蛋白編碼基因的原核表達(dá)質(zhì)粒,在大腸桿菌中誘導(dǎo)表達(dá)、純化重組Fn Cpf1蛋白后,將純化蛋白免疫新西蘭大白兔制備兔抗Fn Cpf1多克隆抗體,間接ELISA法和Western blot分析制備抗體的免疫反應(yīng)性及特異性。并對FnCpf1功能做初步的探討。方法1.以土拉弗朗西絲菌novicida亞種基因組為模板,利用PCR技術(shù)擴(kuò)增Fn Cpf1基因,將其克隆到原核表達(dá)載體p ET-32a(+)中,并用雙酶切和測序驗證。2.將構(gòu)建成功的pET-32a(+)-FnCpf1重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3),IPTG誘導(dǎo)目的蛋白的表達(dá),通過鎳離子金屬螯合磁珠親和純化FnCpf1蛋白。3.將純化蛋白濃縮液與弗氏佐劑充分混合,免疫新西蘭大白兔制備FnCpf1多克隆抗體,并用間接ELISA法檢測兔抗血清效價、Western blot鑒定其特異性。4.構(gòu)建一個插入失活的mcherry熒光報告載體,將其轉(zhuǎn)化至表達(dá)fncpf1蛋白的大腸桿菌感受態(tài)中,用肉眼直接觀察是否出現(xiàn)紅色菌落以判斷是否進(jìn)行了切割、重組。結(jié)果1.擴(kuò)增得到3900bp目的基因片段,克隆到表達(dá)載體pet-32a(+)后,通過雙酶切鑒定大小與預(yù)期一致,測序結(jié)果與genbank一致,證實pet-32a(+)-fncpf1表達(dá)載體構(gòu)建成功。2.可誘導(dǎo)表達(dá)相對分子質(zhì)量約為160kd的目的蛋白,經(jīng)sds-page分析其為可溶性,通過鎳離子金屬螯合磁珠親和純化得到fncpf1純化蛋白,imagelab的體積工具分析結(jié)果表明純度為95%以上,bca法測得超濾濃縮后的純化蛋白濃度為1.4mg/ml。3.用純化蛋白免疫新西蘭大白兔3次后,制備多克隆抗體并檢測兔抗fncpf1抗血清elisa效價達(dá)1:512000,westernblot結(jié)果顯示制備的抗體可較好地與fncpf1蛋白特異性結(jié)合。4.將插入失活的mcherry熒光報告載體轉(zhuǎn)化至表達(dá)fncpf1蛋白的大腸桿菌感受態(tài)中,平板上可見紅色菌落長出,而轉(zhuǎn)化至大腸桿菌dh5α的則未見紅色菌落。結(jié)論1.成功構(gòu)建了fncpf1重組表達(dá)載體,并在原核系統(tǒng)中誘導(dǎo)、表達(dá)和純化fncpf1重組蛋白。2.用純化后的蛋白制備出兔抗fncpf1抗體,效價及特異性均良好,為進(jìn)一步探討cpf1蛋白的生物學(xué)特性打下實驗基礎(chǔ)。3.在表達(dá)FnCpf1的大腸桿菌中,利用CRISPR-Cpf1系統(tǒng)對插入失活的mCherry紅色熒光報告基因進(jìn)行靶向切割并重組,證明了FnCpf1蛋白的核酸酶活性。
[Abstract]:Objective to construct a prokaryotic expression plasmid encoding Cpf1 gene of novicida subspecies in E. coli, and to express the recombinant Fn Cpf1 protein in E. coli, then immunize New Zealand rabbits with purified Fn Cpf1 protein to prepare rabbit anti-Fn Cpf1 polyclonal antibody. Indirect ELISA and Western blot were used to analyze the immuno-reactivity and specificity of the antibody. The function of FnCpf1 is also discussed. Method 1. Using novicida subspecies genome as template, Fn Cpf1 gene was amplified by PCR and cloned into prokaryotic expression vector p ET-32a (), and verified by double enzyme digestion and sequencing. The recombinant plasmid pET-32a ()-FnCpf1 was successfully constructed and transformed into E. coli BL21 (DE3), IPTG-induced target protein expression). FnCpf1 protein was purified by Ni ~ (2 +) metal chelating magnetic beads affinity. 3. FnCpf1 polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein concentrate and Freund's adjuvant. The specificity of the purified protein concentrate was determined by, Western blot of rabbit antiserum titer. An inactivated mcherry fluorescent report vector was constructed and transformed into the competent state of E. coli expressing fncpf1 protein. The presence of red colony was observed directly by naked eye to determine whether it had been cut and recombined. Outcome 1. The target gene fragment of 3900bp was amplified and cloned into the expression vector pet-32a (). The size was confirmed to be consistent with the expected size by double enzyme digestion, and the sequencing results were consistent with that of genbank. It was confirmed that the expression vector pet-32a ()-fncpf1 was constructed successfully. The fncpf1 purified protein was purified by Ni ~ (2 +) metal chelating magnetic beads. The volume tool analysis of imagelab showed that the purity of imagelab was more than 95%, and the protein was soluble by sds-page. The relative molecular weight of 160kd could be induced to express the protein, which was soluble by sds-page and purified by Ni ~ (2 +) metal chelating magnetic beads. The concentration of purified protein after ultrafiltration was 1.4 mg / ml 路3. 3. The concentration of purified protein determined by bca method was 1.4 mg / ml. After immunizing New Zealand rabbits with purified protein for three times, polyclonal antibody was prepared and the titer of elisa in rabbit anti-fncpf1 serum reached 1? 512000. Western blot results showed that the prepared antibody could bind to fncpf1 protein specifically. 4. When the inserted inactivated mcherry fluorescent report vector was transformed into the competent state of E. coli expressing fncpf1 protein, red colony was observed on the plate, but no red colony was found when transformed into E. coli dh5 偽. Conclusion 1. The recombinant expression vector of fncpf1 was successfully constructed and induced, expressed and purified in prokaryotic system. 2. The recombinant protein of fncpf1 was expressed and purified. Rabbit anti-fncpf1 antibody was prepared from purified protein with good titer and specificity, which laid the experimental foundation for further study on the biological characteristics of cpf1 protein. 3. In E. coli expressing FnCpf1, CRISPR-Cpf1 system was used to target cleavage and recombination of the inactivated mCherry red fluorescence reporter gene, which proved the nuclease activity of FnCpf1 protein.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378
本文編號：2445839
[Abstract]:Objective to construct a prokaryotic expression plasmid encoding Cpf1 gene of novicida subspecies in E. coli, and to express the recombinant Fn Cpf1 protein in E. coli, then immunize New Zealand rabbits with purified Fn Cpf1 protein to prepare rabbit anti-Fn Cpf1 polyclonal antibody. Indirect ELISA and Western blot were used to analyze the immuno-reactivity and specificity of the antibody. The function of FnCpf1 is also discussed. Method 1. Using novicida subspecies genome as template, Fn Cpf1 gene was amplified by PCR and cloned into prokaryotic expression vector p ET-32a (), and verified by double enzyme digestion and sequencing. The recombinant plasmid pET-32a ()-FnCpf1 was successfully constructed and transformed into E. coli BL21 (DE3), IPTG-induced target protein expression). FnCpf1 protein was purified by Ni ~ (2 +) metal chelating magnetic beads affinity. 3. FnCpf1 polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein concentrate and Freund's adjuvant. The specificity of the purified protein concentrate was determined by, Western blot of rabbit antiserum titer. An inactivated mcherry fluorescent report vector was constructed and transformed into the competent state of E. coli expressing fncpf1 protein. The presence of red colony was observed directly by naked eye to determine whether it had been cut and recombined. Outcome 1. The target gene fragment of 3900bp was amplified and cloned into the expression vector pet-32a (). The size was confirmed to be consistent with the expected size by double enzyme digestion, and the sequencing results were consistent with that of genbank. It was confirmed that the expression vector pet-32a ()-fncpf1 was constructed successfully. The fncpf1 purified protein was purified by Ni ~ (2 +) metal chelating magnetic beads. The volume tool analysis of imagelab showed that the purity of imagelab was more than 95%, and the protein was soluble by sds-page. The relative molecular weight of 160kd could be induced to express the protein, which was soluble by sds-page and purified by Ni ~ (2 +) metal chelating magnetic beads. The concentration of purified protein after ultrafiltration was 1.4 mg / ml 路3. 3. The concentration of purified protein determined by bca method was 1.4 mg / ml. After immunizing New Zealand rabbits with purified protein for three times, polyclonal antibody was prepared and the titer of elisa in rabbit anti-fncpf1 serum reached 1? 512000. Western blot results showed that the prepared antibody could bind to fncpf1 protein specifically. 4. When the inserted inactivated mcherry fluorescent report vector was transformed into the competent state of E. coli expressing fncpf1 protein, red colony was observed on the plate, but no red colony was found when transformed into E. coli dh5 偽. Conclusion 1. The recombinant expression vector of fncpf1 was successfully constructed and induced, expressed and purified in prokaryotic system. 2. The recombinant protein of fncpf1 was expressed and purified. Rabbit anti-fncpf1 antibody was prepared from purified protein with good titer and specificity, which laid the experimental foundation for further study on the biological characteristics of cpf1 protein. 3. In E. coli expressing FnCpf1, CRISPR-Cpf1 system was used to target cleavage and recombination of the inactivated mCherry red fluorescence reporter gene, which proved the nuclease activity of FnCpf1 protein.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378
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相關(guān)碩士學(xué)位論文 前1條
1 孫菱;新型基因組編輯蛋白FnCpf1的表達(dá)及其功能的初步探討[D];重慶醫(yī)科大學(xué);2017年
，本文編號：2445839
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