摘要
用一组多克隆抗体对马氏甲烷八叠球菌（Ｍｅｔｈａｎｏｓａｒｃｉｎａｍａｚｅｉ）Ｓ－６菌株的基因组ＤＮＡ文库进行了筛选．仅选出ｐ６０Ａ克隆能对马氏甲烷八叠球菌中可发生细胞形态学变化菌株的抗血清发生阳性反应．对ｐ６０Ａ克隆的双链ＤＮＡ进行了序列分析．识别出一开式阅读框架（ｏｐｅｎｒｅａｄｉｎｇｆｒａｍｅＰ，ＯＲＦＰ）．表达的蛋白是ＯＲＦＰ编码的蛋白的３倍，并得到ＯＲＦＰ蛋白的三聚体，在ＳＤＳ－ＰＡＧＥ中表现出整体蛋白的迁移行为．同时，此表达蛋白抗１０％ＳＤＳ，６ｍｏｌ／Ｌ尿素及热处理．基因结构分析表明，ＯＲＦＰ具有与Ｍ．ｂａｒｋｒｉｍｃｒＡ有同源性的核糖体结合位点和一个与甲烷细菌启动子共有序列相似度达７７％的启动子序列．使用参照序列分析表明，由ＯＲＦＰ演绎的氨基酸序列，具有高密度的带电荷的氨基酸和占优势的β－层迭构型．对此表达蛋白作了Ｎｅｕｒｏｓｒｏｒａｃｒａｓｓａ的ｐｏｒｉｎ蛋白抗血清试验，以研究其可能功能．检验了Ｍ．ｍａｚｅｉＳ－６细胞的蔗糖梯度制备物，对此原细胞中的ＯＲＦＰ蛋白作了定位．结果表明，ＯＲＦＰ蛋白可能是一种古细菌Ｐｏｒｉｎ，其在Ｍ．ｍａｚｅｉＳ－６中的表达，可能象大肠杆菌那样与渗透压调节有关．
Methanosarcina mazei S-6 genomic DNA library was screened using a panel of polyclonal antibodies. The clone , termed p60A,reacted only to antiserum against Methanosarcina strains which undergo morphology conversion was chosen for this study. Both DNA strands or clone p60A were sequenced. An open readins frame P (ORF P)was identified. The size of expressed protein is three times as much as that of ORF P. which is confirmed by subeloning and expression of ORF P. The result showed that the expressed protein is a trimer of ORF P protein which has an assedated migration behavior on SDS-PAGE. Also,the expressed protein was resistant to 10% SDS, 6 mol/L urea and heat treatments. The gene structure analysis revealed that ORF P has a putative ribosome binding site homologous to that of M. barkeri mcr A,and a promoter sequence which showed 77 % sequence similarity to promoter consensus sequence of methanogen. The deduced amino acid sequence of ORF P was analyzed with reference sequences, and showed high density of charged amino acids and predominant β-configuration. The expressed protein was tested with antiserum against Neurospora crassa porin to demonstrate its possible function. Sucrose gradient preparations of M. mazei S-6 cells were tacted tolocalize ORF P protein in native cells. The results suggested that ORF P protein is a possible archaebecterial porin and its expression in M. mazei S-6 may be involved in osmotic regulation similar to that in E. coli.
出处
《应用与环境生物学报》
CAS
CSCD
1996年第1期66-78,共13页
Chinese Journal of Applied and Environmental Biology
关键词
古细菌
表达
马氏甲烷八叠菌
结构分析
染色体
archaebacteria
Methanosarcina mazei
DAN sequencing
sequence analysis
Southern blot analysis
sucrose ultrastructure fractionation
