The Bioinformatics Analysis,cloning And Expression Of Mammalian Cell Entry Protein In Nocardia Farcinica And The Research On Its Invasion

Objective To understand the characteristics of mce gene and protein sequence, and to construct the phylogenetic tree by analyzing the homology of sequence, and then to predict secondary structure and the B cell epitopes of six mce proteins in the mce1 operon of nocardia farcinica; To amplify six mce genes from other Nocardia IFM 10152 genome and construct six mce gene expression vectors;To express six mce proteins in the mce1 operon of nocardia farcinica by inducing it,and then to purify the protein which was expressed successfully; The purified protein which was renatured was used to identify the function of invasion. Methods The sequences of mce gene and protein obtained in the NCBI Genebank were analyzed by using the Lasergene software,and the MEGA software was performed to construct the phylogenetic tree; The prediction of B cell epitope of six mce protein was performed by synthesizing BepiPred and the plasticity scheme, the antigenic scheme, the surface accessibility scheme, the hydrophilic scheme of protein and the secondary structure. Six mce genes were amplified by PCR technique, and the expression vector was constructed by gene recombination technique;IPTG was used to induce the recombinant plasmid to express, and the expression was identified by SDS-PAGE after ultrasonic cleavage. The protein was purified by His Bind Protein Purification Kit, and the purified protein was refolded by dialysis method; In this study, we used the protein coated by latex beads to culture with HeLa cells, and then observing the invasion results by transmission electron microscopy after collecting the cells. Results(1)The similarity among six mce gene in the mce1 operon of nocardia farcinica was 40.1%~54.6%, and there was homology of mce protein among different bacteria;(2) All of six mce proteins in the mce1 operon of nocardia farcinica contained a transmembrane structure, and the random coil were the mainly secondary structure; The protein mce1A?mce1D?mce1E contained 6 B cell epitopes, the protein mce1 B contained 7 B cell epitopes, the protein mce1 C contained 2 B cell epitopes,and the protein mce1 F contained 10 B cell epitopes, all of these B cell epitopes located at the C- end of the protein;(3) Six mce genes were successfully amplified, and the recombinant plasmids pET30a-mce1 A ?pET30a-mce1B?pET30a-mce1C?pET30a-mce1D?pET30a-mce1E?pET30a-mce1 F were successfully constructed; The proteins mce1A?mce1C?mce1D?mce1E were successfully induced to express. The expression of mce1 A and mce1 C protein was lower, while the expression of mce1 E and mce1 D protein was in a large amount.All the proteins were expressed in the form of inclusion bodies;(4) Mce1 A, mce1 D and mce1 E protein were purified successfully, and mce1 D and mce1 E proteins were renatured;(5) The mce1 E and mce1 D proteins coated with latex beads were invaded in HeLa cells, the results found that the two proteins were able to enter into the HeLa cells. Conclusion Mce proteins from mycobacterium tuberculosis and nocardia farcinica, which were homology, may evolute from the same gene.Therefore, it was speculated that they had some similar functions. The mce protein,which most of it was out the membrane,might be a membrane protein. and there existed multiple potential B cell epitopes; The expression of mce protein in nocardia farcinica was influenced by gene itself and the vector, but also with the IPTG concentration and inducing time; After obtaining the purified protein, the mce1 D and mce1 E proteins were confirmed to have the function of invasion.