恶性疟原虫红细胞表面膜蛋白1 DBLα区的功能分析
发布时间：2018-09-09 10:58
【摘要】： 本研究根据大肠杆菌密码子组成特点重新合成在GenBank注册的EF158099的1～1179bp序列,即PfEMP1(IT4-var60)DBLα区。另外,利用PCR的方法,成功扩增到优化后的PfEMP1(FCR3S1.2-var1)DBLα区序列,并将优化后的PfEMP1(FCR3S1.2-var1)DBLα区序列分为三段。将两个DBLα区全长基因和PfEMP1(FCR3S1.2-var1)DBLα区的三个基因片段分别在原核系统中表达、纯化,最终获得五个可溶性的重组蛋白。 通过重组蛋白与肝素的亲和试验及GAG抑制试验,分析功能区与肝素/硫化肝素的亲和力,结果表明PfEMP1(IT4-var60)DBLα区对肝素/硫化肝素的亲和力比PfEMP1( FCR3S1.2-var1)D BLα区强,在PfEMP1( FCR3S1.2-var1)D BLα分段表达的三个功能区中,第285～415位氨基酸序列对肝素/硫化肝素的亲和力最强。根据PfEMP1与肝素/硫化肝素结合的分子基础是在其DBLα区含有类似于XBBXXBX或XBBBXXBX结构(B代表碱性氨基酸,如赖氨酸、精氨酸、组氨酸,X代表任意氨基酸),可推测出PfEMP1(FCR3S1.2-var1)DBLα序列中的C R K K K K K L E N L E K Q,S R K G K V R M,D K Q K K Y三个基序可能是被恶性疟原虫寄生的红细胞与红细胞表面的硫化肝素受体结合的关键氨基酸序列。另外,重组的DBLα蛋白与ABO血型抗原亲和试验的结果表明,重组的PfEMP1-DBLα蛋白对血型抗原A、B有特异性的亲和力。该试验结果将为揭示恶性疟原虫的主要致病分子与人体细胞相互作用机制提供很重要的实验依据。
[Abstract]:In this study, the 1~1179bp sequence of EF158099 registered in GenBank, i.e. PfEMP1 (IT4-var60) DBL 伪 region, was resynthesized according to the codon composition of Escherichia coli. In addition, the optimized PfEMP1 (FCR3S1.2-var1) DBL 伪 region sequence was successfully amplified by PCR, and the optimized PfEMP1 (FCR3S1.2-var1) DBL 伪 region sequence was divided into three segments. Two full-length genes of DBL 伪 region and three fragments of PfEMP1 (FCR3S1.2-var1) DBL 伪 region were expressed in prokaryotic system, and five soluble recombinant proteins were obtained. The affinity of PfEMP1 (IT4-var60) DBL 伪 region to heparin / heparin vulcanizate was analyzed by affinity test and GAG inhibition test. The results showed that the affinity of PfEMP1 (IT4-var60) DBL 伪 region to heparin / heparin vulcanizate was stronger than that of PfEMP1 (FCR3S1.2-var1) D BL 伪 region). Among the three functional regions expressed in PfEMP1 (FCR3S1.2-var1) D BL 伪 segment), the amino acid sequence at position 285 was the most compatible with heparin / heparin vulcanizate. According to the molecular basis of the binding of PfEMP1 to heparin / heparin sulfide, its DBL 伪 region contains a structure similar to that of XBBXXBX or XBBBXXBX (B represents basic amino acids such as lysine, arginine, arginine). Histidine X represents any amino acid). It can be inferred that the three C R K K K K K L E N L E K QN S R K G K V R Med K Q K K Y motifs in PfEMP1 (FCR3S1.2-var1) DBL 伪 sequence may be erythrocytes and erythrocytes parasitized by Plasmodium falciparum (Plasmodium falciparum). The surface of the sulphide heparin receptor binds to the key amino acid sequence. In addition, the result of affinity test of recombinant DBL 伪 protein with ABO blood group antigen showed that the recombinant PfEMP1-DBL 伪 protein had specific affinity to blood group antigen Anb. The results will provide an important experimental basis for revealing the interaction mechanism between the main pathogenic molecules of Plasmodium falciparum and human cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R382
本文编号：2232181
[Abstract]:In this study, the 1~1179bp sequence of EF158099 registered in GenBank, i.e. PfEMP1 (IT4-var60) DBL 伪 region, was resynthesized according to the codon composition of Escherichia coli. In addition, the optimized PfEMP1 (FCR3S1.2-var1) DBL 伪 region sequence was successfully amplified by PCR, and the optimized PfEMP1 (FCR3S1.2-var1) DBL 伪 region sequence was divided into three segments. Two full-length genes of DBL 伪 region and three fragments of PfEMP1 (FCR3S1.2-var1) DBL 伪 region were expressed in prokaryotic system, and five soluble recombinant proteins were obtained. The affinity of PfEMP1 (IT4-var60) DBL 伪 region to heparin / heparin vulcanizate was analyzed by affinity test and GAG inhibition test. The results showed that the affinity of PfEMP1 (IT4-var60) DBL 伪 region to heparin / heparin vulcanizate was stronger than that of PfEMP1 (FCR3S1.2-var1) D BL 伪 region). Among the three functional regions expressed in PfEMP1 (FCR3S1.2-var1) D BL 伪 segment), the amino acid sequence at position 285 was the most compatible with heparin / heparin vulcanizate. According to the molecular basis of the binding of PfEMP1 to heparin / heparin sulfide, its DBL 伪 region contains a structure similar to that of XBBXXBX or XBBBXXBX (B represents basic amino acids such as lysine, arginine, arginine). Histidine X represents any amino acid). It can be inferred that the three C R K K K K K L E N L E K QN S R K G K V R Med K Q K K Y motifs in PfEMP1 (FCR3S1.2-var1) DBL 伪 sequence may be erythrocytes and erythrocytes parasitized by Plasmodium falciparum (Plasmodium falciparum). The surface of the sulphide heparin receptor binds to the key amino acid sequence. In addition, the result of affinity test of recombinant DBL 伪 protein with ABO blood group antigen showed that the recombinant PfEMP1-DBL 伪 protein had specific affinity to blood group antigen Anb. The results will provide an important experimental basis for revealing the interaction mechanism between the main pathogenic molecules of Plasmodium falciparum and human cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R382
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