Cloning, Recombinant Expression, Purification And Identification Immunological Characters Of Ferritin Gene From Echinococcus Granulosus(Chinese Mainland Strain)

Object To clone Chinese mainland strain ferritin gene from RNA of echinococcus granulosus and construct prokaryotic expressed recombinant plasmid. Then to express, purify and identify the immunological character of the recombinant ferritin. Providing valuable candidate gene to develop vaccine for preventing echinococcus granulosus.Method (1)Total RNA was extracted from protoscoleces of cysts of human. The specific primers were designed according to published nucleotide sequence of NCBI/Genbank database. The ferritin gene of Echinococcus granulosus was amplified by RT-PCR. (2)The ferritin gene was insert into pGEM-T vector and transfer into E.coil JM109. Then sending the recombinant plasmid Eg.ferritin/pGEM-T to company for sequencing. (3)Using DNAman software and NCBI/BLAST database to analyze homology of ferritin gene of many species and using DNAstar, Biosun biological softwares to predict second structure and antigen peptide of Eg.ferritin, using SWISSMODEL to imitate 3D structure of Eg.ferritin. (4) Construct the first expressed recombinant plasmid: a. The ferritin gene was subcloned into high-level expression vector pGEX-6P-1 and was transferred into E.coil BL21. Then Eg.ferritin/pGEX-6P-1 was expressed fusion protein Eg.ferritin/GST by IPTG. Induction. Using GSTTMFF affinity chromatography to purify Eg.ferritin/GST. b. Cutting Eg.ferritin/GST band of SDS-PAGE to immunize BABL/c mice and to research its immunological characters by western-blot. (5) Construct the second expressed recombinant plasmid: a. Eg.ferritin gene was subclone into new expression vector pET-28a. Then express and purify the recombinant protein Eg.ferritin By His-bind affinity chromatography with Ni2+. b. Using purified Eg.ferritin to immunize ICR mice and obtaining the specific sera to test biological activity of Eg.ferritin by western-blot and ELISA.Result (1) A gene sequence of 562bp has been amplified by RT-PCR. (2) Comparing 390bp of the DNA and deduced amino acid sequence with the published ferritin gene sequence of Echinococcus granulosus to revealed 97.7% homology and average homology of different species are 40.79%. Using softwares to predict: molecule weight of Eg.ferritin is 16.7kD. And it has 68 Nonpolar amino acids and has 9 antigen peptide such as 7N-12E, 36H -43V, 55S-62H, 69Q-76R, 82A-89E, 102I-107E, 117A–124S, 129L–136T.(3)Construct genetic engineering strain Eg.ferritin/pGEX-6P-1/BL21 and express fusion protein Eg.ferritin/GST. But the fusion protein cannot be purifying because it is inclusion body. Eg.ferritin/GST could be recognized by antibody sera of rabbit immunized with Echinococcus granulosus and special sera from mice immunized with Eg.ferritin/GST band of SDS-PAGE and The special sera can also recognized native antigen cyst fluid and protoscoles of Echinococcus granulosus about 19kD band through Western-blot. (4)Construct recombinant expressive vector Eg.ferritin/pET-28a and express, purify the recombinant protein Eg.ferritin Successfully. This purified Eg.ferritin and native antigen cyst fluid and protoscoles of Echinococcus granulosus can be recognized by specific sera of mice immunized with purified Eg.ferritin and These recognized protein band have resemble position about 19kD. Through ELISA we find IgG level of sera of the group mice, which is immunized by Eg.ferritin, is higher than control group conspicuously on the same period (P<0.01).Conclusions (1)Cloning ferritin gene of Chinese mainland strain from Echinococcus granulosus and constructing gene engineering strain Eg.ferritin/pGEM-T/JM109 successfully. (2)Using softwares to predict Eg.ferritin structure and antigen peptide to provide some theoretical bases for practicing my experiment and selecting some valuable antigen peptide. (3)Construct gene engineering strain Egferritin/pGEX-6P-1/BL21 successfully and express Eg.ferritin/GST about 42kD. Because the fusion protein is inclusion body so it cannot be purified. (4)Construct gene engineering strain Eg.ferritin/pET-28a/BL21(DE3)plysS and express, purify Eg.ferritin of 19kD. The Eg.ferritin have higher immunogenicity and may be a new vaccine candidate of Echinococcus granulosus .