| In recent years,the incidence of non gonococcal urethritisthe(NGU)caused by Chlamydia trachomatis(C.t)is increasing.in the United States,its incidence ranked the top five in the common diseases;in China,the outpatient detection rate was 9.9%,the most majority are young people.However,in recent years,drug-resistant strains make it difficult to treat Ct infection.It is gratifying to note that many of the current clinical literature and studies have shown that Chlamydiaphage can be used as a new antibacterial therapy for the treatment of Ct infection.Research earlier have successfully expressed and purified the capsid protein Vp1,the largest and most important capsid protein of phiCPG1,the Vp1 was found that it could inhibit the growth of Ct,and the inhibition rate of Ct type E was 78%.Vp1 has 1659 bp in length and it is highly conserved,while there are two special regions in the amino acid sequences of Vp1,which are called IN5 loop and INS loop,located in the 216-299 and 462-467 amino acid sequences,respectively.The IN5 loop forms a mushroom like protrusion on the surface of Chlamydiaphage capsid protein,and the IN5 loop plays a key role in the recognition of Chlamydiaphage and chlamydia.Spiroplasma virus,SpV4 and Chlamydiaphage all belong to the the family Parvoviridae and there are also mushroom like projections on the surface of SpV4 capsid protein.There is a mainly hydrophobic cavity formed by hydrophobic amino acid residues at the distal surface of the protrusion,the hydrophobic cavity are involved in the recognition process of phage and host.In this study,we have changed the hydrophobicity of the Vp1 protein of Chlamydiaphage phiCPG1 as well as cloned and expressed the IN5 part of the Vp1 protein.Then,the purified proteins were co-cultured with the Ct and compared the inhibit rate.The study provides a reference value for the later exploration of the optimal design of the Vp1 protein.[Objective] The aim is to obtain Vp1 protein with higher inhibitory rate to Chlamydia trachomatis,and to provide a new clinical treatment thoughts.[Method] The IN5 region of the Vp1 protein was cloned and amplified by using the capsid protein Vp1 of Chlamydia phage phiCPG1 as template to construct the expression plasmid;Comparing the amino acid sequence of capsid protein Vp1 of SpV4 and phiCPG1 to found the position that hydrophobic amino acids on SpV4 corresponding to where they are on the phiCPG1 Vp1.Then,we designed to change the hydrophobicity of amino acids,the mutant Vp1,Vp1m1;Through bioinformatics software PredictProtein analysis of key binding sites of Chlamydiaphage phiCPG1 and host,then we changed of hydrophobic amino acids to construct mutant Vp1,Vp1m2.Expressing the three proteins of IN5、Vp1m1 and Vp1m2 and identifying the proteins by SDS-PAGE and Western-blot.The purified protein and the controls groups was co-cultured with Ct,after 48 h,indirect immunofluorescence was used to count the inclusion bodies to compare the inhibition rate between the three proteins.[Results] The proteins of Vp1m1,Vp1m2 and IN5 part of Vp1 protein were successfully expressed and purified.The inhibition rates of Vp1m1,Vp1m2 and IN5 on the growth of Ct were 69.59%,89.07% and 54.50%,respectively.[Conclusion] The protein Vp1m2 which has a higher inhibition rate than the original Vp1 protein against Chlamydia trachomatis.This study provides clues for searching the dominant functional regions of Vp1 protein and the mechanism of the interaction between phage and host.At the same time,it also provides a preliminary exploration experience to optimize the design of Vp1 protein with higher inhibition rate and provide a new clinical treatment thoughts... |
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