Studies On Expression Of Human Lysozyme Gene HLYZ In Pichia Pastrois And Mouse Mammary Glands And Its Biological Activity

Lysozyme is a group of small molecule basic peptide, which was encoded by genome and synthesized by ribosome, and produced by plants, insects, amphibians and mammals including human beings. Lysozymes characterize small molecular weight, weak antigenicity, strong stability, unique antibacterial mechanism, hardly inducing the drug resistance.etc. Besides the antibacteria activity with broad spectrum, lysozymes also inhibit the development of viruses and cancer cells. They have been a new kind of biological preparation to cope the increasingly serious problemsof drug resistance. Due to the high cost, low yield and the complicated process in extraction lysozymes in natural stock, this research aimed to produce human lysozyme through technique for gene engineering and to explore new methods on large-scale preparation of human lysozyme.The gene of human lysozyme was ampified by RT-PCR from the issue of human placenta. The primers was designed according to the sequence offered by Gene Bank. The research of cloning, sequencing and analyzing this gene suggested that the gene contains 390bp bases, and encodes 130 amino acid residues. The molecular weight is 14633.64Da, Isoelectric point is 8.67. Under pH value 7.0 conditions, the protein has positive charge. This protein has very comlex secondary structure with littleα-structure or coil, whileβ-structures are a lot.α-structure mainly concentrate in N-terminio andβ-structures in C-terminio. The amphipathic properties of this protein is very high, with several flexibile domains in them.The gene consistents with the sequence repoted by Gene Bank.The recombinant of human lysozyme was secreting expressed in Pichia Pastrois expressing system. The GS115 strain was selected to be the host of eukaryotic expression.The process of secreting expression of the recombinant of human lysozem was introducted though the ultilization of theα-factor, while the vector pPIC9k was the vetor of eukaryotic expression. An expressing system consisted of Pichia Patrois GS115, expressing vector pPIC9k and the gene of human lysozyme hLYZ was constructed, from which 16 strains of positive clones were selected trough G418 resistance, and nutrition selecting. 1 strain with high activity of antibacteria was selected through bacteriostatic activity selecting in vitro. In the condition that BMGY : BMMY made 1 : 1, 28℃, concentration of methanol was lower than 1%, the strain was introduced expression. The supernatant was concentrated and examined the activity of antibacterial. This result suggested that The recombinant of human lysozyme was able to inhibit and kill E.coli ( ATCC25922 ) , Streptococcus (ATCC55121), Bacillus paratyphosus(S.C500), Bacillus cereus (CMCC(B)63301) and Candida albicans (ATCC10231) in defferent degree. The activity of recombinant of human lysozyme expressed in Pichia Pastrois was calculated and the record was 4 677.72 U·mL-1。The recombinant of human lysozyme was also expressed in the mammary of mice. The vector pcDNA3.1 was selected to be the vector of this eukaryotic expression. The expressing vector which was named pcDNA3.1-hLYZ was constructed and transfected into mice and then expressed. The milk and breast issue of the mice were corllected to identify the expression of gene and protein by means of RT-PCR , immunohistochemisty and Western blotting. The recombinant of human lysozyme was able to inhibit and kill E.coli(ATCC25922)and Micrococcus lysodeikticus, that was identified through bacteriostatic activity examination. The activity of recombinant of human lysozyme expressed in the milk of mice was calculated and the record was 4011 U?mL-1。...