惡性瘧原蟲紅細胞表面膜蛋白1 DBLα區(qū)的功能分析
發(fā)布時間：2018-09-09 10:58
【摘要】： 本研究根據(jù)大腸桿菌密碼子組成特點重新合成在GenBank注冊的EF158099的1～1179bp序列,即PfEMP1(IT4-var60)DBLα區(qū)。另外,利用PCR的方法,成功擴增到優(yōu)化后的PfEMP1(FCR3S1.2-var1)DBLα區(qū)序列,并將優(yōu)化后的PfEMP1(FCR3S1.2-var1)DBLα區(qū)序列分為三段。將兩個DBLα區(qū)全長基因和PfEMP1(FCR3S1.2-var1)DBLα區(qū)的三個基因片段分別在原核系統(tǒng)中表達、純化,最終獲得五個可溶性的重組蛋白。 通過重組蛋白與肝素的親和試驗及GAG抑制試驗,分析功能區(qū)與肝素/硫化肝素的親和力,結果表明PfEMP1(IT4-var60)DBLα區(qū)對肝素/硫化肝素的親和力比PfEMP1( FCR3S1.2-var1)D BLα區(qū)強,在PfEMP1( FCR3S1.2-var1)D BLα分段表達的三個功能區(qū)中,第285～415位氨基酸序列對肝素/硫化肝素的親和力最強。根據(jù)PfEMP1與肝素/硫化肝素結合的分子基礎是在其DBLα區(qū)含有類似于XBBXXBX或XBBBXXBX結構(B代表堿性氨基酸,如賴氨酸、精氨酸、組氨酸,X代表任意氨基酸),可推測出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三個基序可能是被惡性瘧原蟲寄生的紅細胞與紅細胞表面的硫化肝素受體結合的關鍵氨基酸序列。另外,重組的DBLα蛋白與ABO血型抗原親和試驗的結果表明,重組的PfEMP1-DBLα蛋白對血型抗原A、B有特異性的親和力。該試驗結果將為揭示惡性瘧原蟲的主要致病分子與人體細胞相互作用機制提供很重要的實驗依據(jù)。
[Abstract]:In this study, the 1~1179bp sequence of EF158099 registered in GenBank, i.e. PfEMP1 (IT4-var60) DBL 偽 region, was resynthesized according to the codon composition of Escherichia coli. In addition, the optimized PfEMP1 (FCR3S1.2-var1) DBL 偽 region sequence was successfully amplified by PCR, and the optimized PfEMP1 (FCR3S1.2-var1) DBL 偽 region sequence was divided into three segments. Two full-length genes of DBL 偽 region and three fragments of PfEMP1 (FCR3S1.2-var1) DBL 偽 region were expressed in prokaryotic system, and five soluble recombinant proteins were obtained. The affinity of PfEMP1 (IT4-var60) DBL 偽 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 偽 region to heparin / heparin vulcanizate was stronger than that of PfEMP1 (FCR3S1.2-var1) D BL 偽 region). Among the three functional regions expressed in PfEMP1 (FCR3S1.2-var1) D BL 偽 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 偽 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 偽 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 偽 protein with ABO blood group antigen showed that the recombinant PfEMP1-DBL 偽 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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R382
本文編號：2232181
[Abstract]:In this study, the 1~1179bp sequence of EF158099 registered in GenBank, i.e. PfEMP1 (IT4-var60) DBL 偽 region, was resynthesized according to the codon composition of Escherichia coli. In addition, the optimized PfEMP1 (FCR3S1.2-var1) DBL 偽 region sequence was successfully amplified by PCR, and the optimized PfEMP1 (FCR3S1.2-var1) DBL 偽 region sequence was divided into three segments. Two full-length genes of DBL 偽 region and three fragments of PfEMP1 (FCR3S1.2-var1) DBL 偽 region were expressed in prokaryotic system, and five soluble recombinant proteins were obtained. The affinity of PfEMP1 (IT4-var60) DBL 偽 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 偽 region to heparin / heparin vulcanizate was stronger than that of PfEMP1 (FCR3S1.2-var1) D BL 偽 region). Among the three functional regions expressed in PfEMP1 (FCR3S1.2-var1) D BL 偽 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 偽 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 偽 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 偽 protein with ABO blood group antigen showed that the recombinant PfEMP1-DBL 偽 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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R382
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