Secreted Expression Of Bos Taurus Lysozyme-1 In Pichia Pastoris And Biological Activities Detection

Lysozyme is an important defensive factor presented in the humoral and tissues of human and other animals. Therefore, it plays a significant role in the non-specific immunity. It also has many other pharmacological functions, such as anti-bacterium, anti-inflammation, promoting organism recovery. That is why, it is widely used in food and feed industries, scientific research andmedical clinical treatments. As a c-type lysozyme in a kind of dairy cows, Bos taurus lysozyme 1(LYZ1) not only has the similar biological effects, but also has homology advantage in the treatment of dairy cow endometritis and mastitis which caused by bacterium. LYZ1 should be widely used in clinical appliance. However, the complicated steps of extracting lysozyme from organism and the high prices maked it hardly to be used widely in clinical applications. Therefore, in this study the Escherichia coli and Pichia pastoris expression system was constructed in order to obtain the recombinant LYZ1with high activition and replace the natural lysozymes. Details of the study are as follows:(1)Based on the gene sequences encoding LYZ1 which was registered in GenBank, selecting the preference codons of E.coli and Pichia pastoris, LYZ1 was designed and synthesized.(2)The EcoR I and Xho I restriction sites were contained at the upstream and downstream of the LYZ1, respectively. A his-tag was introduced to simplify the purification procedures later on. Then the LYZ1 gene was cloned into expression vector pET-32a. The constructed recombinant plasmid pET-32a-LYZ1 which identified by PCR identification, both restriction analysis and sequencing was subsequently transformed to E.coli BL21(DE3). Obtained LYZ1 engineered strain BL21/pET-32a- LYZ1. Under induction of IPTG, engineered strain BL21/pET-32a-LYZ1 expressed protein, with a relative molecular mass of about 32 Ku, mainly existed in a form of inclusion body and contained about 70% of total somatic protein,and was provided with specific immunogenicity. After purification, the recombinant protein reached a purity of more than 95%. We immunized BALB/c rats with the recombinant proteins in order to preparate single factor serum which was expect to identify the recombinant LYZ1 was expressed in Pichia pastoris(3)XbaⅠrestriction site was added at downstream of the gene LYZ1,then the LYZ1 was cloned into the Pichia pastoris expression vector pPICZα-A to construct the recombinant plasmid pPICZα-A -LYZ1. The SacⅠlinearized plasmid pPICZαA-LYZ1 was transformed into Pichia pastoris strain GS115 by electroporation, Recombinant strains were screened on YPDS plates containing Zeocin and identified by PCR. Recombinant Pichia GS115/pPICZαA-LYZ1 was induced by methanol for 60 hours, SDS-PAGE analysis showed that recombinant protein was expressed in soluble form with molecular weight approximately 16 Ku and Western Blotting demonstrated good immunogenicity. Expression of the supernatant concentrated by ultrafiltration tube, over-his-tag column purified recombinant protein was measured in the supernatant concentration of 218 mg/L. Activity was detected by Micrococcus lysodeikticus(ML). Activity of recombinant protein was 2842 U/mL , and the specific activity was approximately 13800 U/mg in the supernatant. The effect of vitro bacteriostasis was detected. The recombinant LYZ1 had better antibacterial activity to Staphylococci and Bacterium coli. It is inferio to Streptococcus mastitidis, Streptococcus dysgalactiae and Streptococcus uberis.Based on these data, in this study, Prokaryotic expression vector pET-32a-LYZ1 and eukaryotic expression vector pPICZαA-LYZ1 were successfully constructed. Then, recombinant protein LYZ1 were successfully expressed in the recipient bacteria E.coli BL21(DE3) and Pichia pastoris yeaststrain GS115. The recombinant protein was expressed in soluble form having antibacterial activity. The work provides the basis for further application of LYZ1.