Construction Of Chimeric Prokaryotic Expression Vector Of Cp23Gene From Cryptosporidium Parvum Into Slp Gene From Lactobacillus Brevis

S-layer protein is usually located in the outer surface of cell wall or cell membrane of bacteria and archaea with single-molecule and Lattice-like structure. These proteins has a unique inherent properties which easily ruptured by chemicals, than protein crystals can be reassembled into the original lattice. Its function is maintain the shape of the bacterial cells, combined with the high molecular enzymes, adhesion and invasion the target cells. It is an important structure for the bacteria to adapt to the environment. Some Lactobacillus S-layer protein with molecular weight between25-71KDa is currently the smallest known S-layer protein.Cryptosporidium parvum parasitic in humans and mammals which can cause cryptosporidiosis. The pathogenic and immune-related antigen protein exist in its every complex life cycle.3D kinds of promising vaccine candidate molecules have been reported, such as GP900, CSL, Cp23, Cp15/40/60, Cp12and Cp21.Because of the conservative and stronger immune activity, the Cryptosporidium sporozoite surface antigen which encoded by Cp23gene sequence is considered as the best target antigen of Cryptosporidium parvum in immunodiagnosis and vaccines developed by most scholars.This study uses molecular biology techniques to construct the chimeric expression vector with surface antigen Cp23gene and the S-layer protein gene of Lactobacillus brevis. Lay the foundation for the achievement of the gene fusion joint development of the vaccine vector. The experimental results are as follows:1Survey and identification the S-layer protein and sip gene of the lactic acid bacterias by SDS-PAGE and PCR respectively in protein and nucleic acid level. Detect the size of the S-layer protein of Lactobacillus brevis is approximately47kDa.2Analyzed the slp gene sequences of Lactobacillus brevis through the tools such as Genetyx and Bioinf. The sequencing results showed that the effective nucleotide sequence of Lactobacillus brevis is1263bp which deduced421amino acids, and determine the outergcnc into the exact sites.3The recombinant plasmid pET30a-slp-P23was successfully constructed applicated the recombinant DNA technology with exogenous CP23gene and slp gene. 4The recombinant plasmid pET30a-slp-P23was transferred into E.coil BL21which was induced by IPTG. Analysised by SDS-PAGE and Western blotting, the result showed positive.