放线共生放线杆菌菌毛重组蛋白的提取及纯化

J4 ›› 2011, Vol. 37 ›› Issue (2): 370-374.

放线共生放线杆菌菌毛重组蛋白的提取及纯化

李毅¹|郭学军²|杨涛³|冯书章²|孙宏晨¹

1.吉林大学口腔医院儿童口腔科|吉林长春 130021;2.中国军事科学院军事兽医研究所分子细菌学研究室,吉林长春 130062;3.吉林省长春市儿童医院儿童口腔科|吉林长春 130051

收稿日期:2010-09-04 出版日期:2011-03-28 发布日期:2011-03-28
通讯作者: 孙宏晨(Tel:0431-88796038,E-mail:liyikq2004@yahoo.com) E-mail:liyikq2004@yahoo.com
作者简介:吉林省科技厅科研基金资助课题(201015202)
基金资助:
吉林省科技厅科研基金资助课题(201015202)

Extraction and purification of |recombinant protein LTB-FAPof fimbria of Actinobacillus actinomycetemcomitans

LI Yi¹,Guo Xue-jun²,Yang Tao³,Feng Shu-zhang²,Sun Hong-chen ¹

1.Department of Pediatric Dentistry,Stomatology Hospital,Jilin University,Changchun 130021,China;2.Department of Molecular Bacteriology,Institute of |Military Veterinary Sciences,Academy of Military Medical Sciences,Changchun 130062,China;3.Department of Pediatric Dentistry,Changchun Children Hospital,Changchun 130051,china

Received:2010-09-04 Online:2011-03-28 Published:2011-03-28

摘要/Abstract

摘要：

［摘要］目的:对大肠杆菌表达的放线共生放线杆菌菌毛重组蛋白LTB-FAP进行提取和纯化，为其用于牙周病的治疗提供理论依据。方法: 将重组质粒pET28a/LTB和pET28a/LTB-FAP分别转化到大肠杆菌中进行表达，通过超声裂解法获得以包涵体形式表达的重组外源蛋白LTB-FAP；用2,4,6和8 mol·L^-1尿素缓冲液对重组蛋白进行洗涤、溶解；然后用阴离子交换和凝胶过滤柱层析对重组蛋白进行纯化,采用薄层扫描仪对蛋白纯度进行测定。结果:目的包涵体蛋白经过3次4 mol·L^-1尿素洗涤，6 mol·L^-1尿素溶解后，经薄层扫描可获得纯度为60%的目的蛋白；经离子交换层析纯化，蛋白纯度可达约75%；Sepharose 6B行凝胶过滤层析获得了纯度可达90%的重组蛋白LTB-FAP。结论: 阴离子交换和凝胶过滤柱层析的方法可提取、纯化重组蛋白LTB-FAP,获得的LTB-FAP可以用于免疫机体。

关键词: 放线共生放线杆菌；LTB-FAP；提取；纯化

Abstract:

Abstract:Objective To extract and purify the rombinant protein LTB-FAP of fimbria of Actinobacillus actinomy cetecomitans which can be expressed in E.coli. and lay a foundation for its application in the treatment of periodontal diseases.Methods The constructed recombinant plasmid of pET28a/LTB and pET28a/LTB-FAP were transformed into E.coli and the inclusion body of recombinant protein LTB-FAP was expressed. The recombinant protein was washed and dissolved by 2,4,6 and 8 mol·L^-1 urea;and then the dissolved protein was purified by both anion exchange chromatography and gel filtration chromatography;the concentration ofthe purified protein LTB-FAP was measured by thin layer chromatogram scanner.Results The inclusion body of the expressed protein was washed 3 times by 4 mol·L^-1 urea solution and dissloved in 6 mol·L^-1 urea solution,and 60% of the LTB-FAP protein could be gotten. The purity of the recombinant protein LTB-FAP purified in this experiment by anion exchange chromatography was about 75%;the puity of recombinant protein LTB-FAP was 90% by gel filtration chromatography. Conclusion The anion exchange chromatography and gel filtration chromatography can extract and purify the expressed recombinant protein LTB-FAP which can be used to immunize animal bodys.

Key words: Actinobacillus actinomycetemcomitans;LTB-FAP;extraction;purification

中图分类号:

R379.1

李毅|郭学军|杨涛|冯书章|孙宏晨 .
放线共生放线杆菌菌毛重组蛋白的提取及纯化[J]. J4, 2011, 37(2): 370-374.

LI Yi,Guo Xue-jun,Yang Tao,Feng Shu-zhang,Sun Hong-chen . Extraction and purification of |recombinant protein LTB-FAPof fimbria of Actinobacillus actinomycetemcomitans[J]. J4, 2011, 37(2): 370-374.