本文選題：豬CR1-like　切入點：生物信息學(xué)　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：目的：利用巴斯德畢赤酵母(Pichia pastoris)表達(dá)系統(tǒng),對豬紅細(xì)胞CR1-1ike的補(bǔ)體調(diào)控蛋白區(qū)域(complement control protein domain, CCP)進(jìn)行串聯(lián)表達(dá),為進(jìn)一步深入研究豬CR1-like的作用機(jī)制奠定基礎(chǔ)。方法：對豬CR1-like的CCPs氨基酸序列進(jìn)行生物信息學(xué)分析,篩選出CCPs的基因序列,密碼子優(yōu)化后人工合成CCPs基因序列；將合成的基因序列連入表達(dá)載體pwPICZalpha中,利用Pichia pastoris對其進(jìn)行重組表達(dá), SDS-PA GE和Western blotting檢測表達(dá)上清；利用Ni-NTA Resin柱和強(qiáng)陰離子交換柱對重組蛋白進(jìn)行純化；采用激光共聚焦顯微鏡觀察重組蛋白與經(jīng)兔血清致敏菌株或未致敏菌株的免疫粘附反應(yīng),及豬CRl-like單克隆抗體先與重組蛋白反應(yīng),再與豬紅細(xì)胞共孵育后的免疫阻斷反應(yīng)。結(jié)果：生物信息學(xué)分析結(jié)果顯示,豬CRl-like基因含有19個CCP,并選定3-6和8-11兩段CCPs基因序列進(jìn)行表達(dá)；SDS-PAGE和Western blotting結(jié)果顯示,表達(dá)上清中存在所預(yù)期的目的蛋白,說明重組酵母構(gòu)建成功；表達(dá)上清經(jīng)純化濃縮后,獲得了分別濃度為0.828 mg/mL和0.945mg/mL的兩種重組蛋白；免疫熒光結(jié)果顯示,重組蛋白可與致敏短小芽孢桿菌發(fā)生粘附,并能夠阻斷CRl-1ike單克隆抗體與豬紅細(xì)胞的結(jié)合。結(jié)論：本試驗成功獲得兩株可穩(wěn)定遺傳表達(dá)豬CR1-like CCPs的Pichia pastoris菌株；重組蛋白表現(xiàn)出補(bǔ)體結(jié)合活性,具有類免疫粘附受體功能。
[Abstract]:Aim: to express the complement control protein domain (CCPs) of porcine erythrocyte CR1-1ike by using Pichia pastoris expression system. Methods: the CCPs amino acid sequence of porcine CR1-like was analyzed by bioinformatics, the gene sequence of CCPs was screened out, and the CCPs gene sequence was synthesized after codon optimization. The synthesized gene sequence was inserted into the expression vector pwPICZalpha and expressed by Pichia pastoris. The expression supernatant was detected by SDS-PA GE and Western blotting, and the recombinant protein was purified by Ni-NTA Resin column and strong anion exchange column. Laser confocal microscopy was used to observe the immune adhesion reaction between recombinant protein and strain sensitized or not sensitized by rabbit serum, and the monoclonal antibody against porcine CRl-like was first reacted with recombinant protein. Results: the results of bioinformatics analysis showed that the porcine CRl-like gene contained 19 CCPs, and selected 3-6 and 8-11 segments of CCPs gene sequence to express SDS-PAGE and Western blotting. The expression of the target protein in the supernatant indicated that the recombinant yeast was successfully constructed, and two recombinant proteins with concentrations of 0.828 mg/mL and 0.945mg/mL were obtained after purification and concentration of the supernatant. The recombinant protein could adhere to the sensitized Bacillus pumilus and block the binding of CRl-1ike monoclonal antibody to porcine erythrocyte. Conclusion: in this experiment, two stable Pichia pastoris strains expressing porcine CR1-like CCPs were successfully obtained. The recombinant protein exhibits complement binding activity and has the function of immune adhesion receptor.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q786;S852.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 趙孟春;李宏全;段瑞旭;白瑞;;紅細(xì)胞免疫的物質(zhì)基礎(chǔ)及影響因素[J];浙江畜牧獸醫(yī);2007年03期

2 劉高科;何莉;楊永濤;汪正清;;人補(bǔ)體受體1型活性片段SCR15-18在畢赤酵母中的表達(dá)、純化及活性鑒定[J];第三軍醫(yī)大學(xué)學(xué)報;2009年06期

3 王玲;蒲萬霞;扎西英派;索朗斯珠;孟曉琴;李蕓;程勝利;孟聚誠;華蘭英;;酵母活性物質(zhì)對斷乳仔豬紅細(xì)胞免疫功能的影響[J];動物醫(yī)學(xué)進(jìn)展;2007年08期

4 華慧,周思翔,王正榮;外源基因在巴斯德畢赤酵母中的表達(dá)[J];國外醫(yī)學(xué).生物醫(yī)學(xué)工程分冊;2003年03期

5 左之才;崔恒敏;甘夢;彭西;鄧俊良;;感染豬放線桿菌(Ⅰ型)對豬紅細(xì)胞免疫功能的影響[J];東北農(nóng)業(yè)大學(xué)學(xué)報;2012年12期

6 楊紹俊;張璇;汪正清;;人補(bǔ)體受體1型SCR1-3功能域基因的克隆表達(dá)及生物活性鑒定[J];免疫學(xué)雜志;2012年01期

7 黃科,曹家樹,吳秋云,溫慶放;生物信息學(xué)[J];情報學(xué)報;2002年04期

8 范翠英;馮利興;樊金玲;果德安;劉璇;;重組蛋白表達(dá)系統(tǒng)的研究進(jìn)展[J];生物技術(shù);2012年02期

9 王瑞剛,王水平;外源蛋白表達(dá)系統(tǒng)及利用植物表達(dá)外源蛋白的特點與優(yōu)勢[J];生物學(xué)通報;2003年01期

10 王旭東，饒家榮，李正甫，朱文斌，楊紅華，周述成，李天祥，張世洪;11類哺乳類和鳥類動物紅細(xì)胞免疫功能的對比研究[J];畜牧獸醫(yī)學(xué)報;1995年06期

相關(guān)碩士學(xué)位論文 前1條

1 馮帆;新基因CCP22功能的初步研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年

，

本文編號：1653552