Cloning And Expression For S Gene Of TGEV And Generation Of Transgenic Mouse

The mammary glands of transgenic animals are an ideal site for producing complex bioactive proteins. This technology overcomes the limitations of conventional and recombinant production systems for recombinant proteins. Mammary gland bioreactor which can provide protection against enteric virus infection for animal immunized by secreting virus antigen protein into their milk is a promising expression system to produce mammalian viral vaccines in an economic and scalable manner. To investigate the feasibility of mammary gland bioreactor producing efficacious mammalian viral vaccine, in this study, the expression of the recombinant spike (S) protein from swine-transmissible gastroenteritis virus (TGEV) was tested in mouse mammary cells in vitro and in vivo, and transgenic mice were generated via seminiferous tubules injection.S gene of TGEV TSX strain was amplified by RT-PCR and recombinant PCR. The amplified DNA fragments were purified from agrose gel, subsequently cloned into pGEM-T vector, and then transformed into E.coli DH5α. The positive clones were named pGEM-S, which were identified by restriction endonuclease digestion and sequencing analysis. The whole 4 350 bp S gene was amplified, which encoded 1 449 amino acids. The N-end initiative 16 amino acids were signal peptide and the latter 1 433 amino acids were mature protein. Comparison with nine other S gene of TGEV strains registered in GenBank showed 95%-98% homology in DNA sequence and 95%-98% homology in protein. Phylogenetic tree based on S gene and deduced amino acid sequence of 10 different strains of TGEV showed that TSX had closer relationships with Miller, PUR46-MAD, TH-98, TS, and HN2002. Moreover, there were some differences in the antigenic site between TSX and other strains of TGEV, but the main antigenic site doesn't change. Therefore, the study provides a good candidate gene for the research of gene engineering vaccine.S gene was cloned into middle of 5' end regulatory element and 3' end regulatory element of the bovine beta-casein (β-casein, CSN2)promoter, the recombinant plasmid pBS was constructed. We obtained bs fragment which was 7.3 kb with SacⅠ/MluⅠdigestion, then was cloned directionally into expression vector pEGFP-C1. The mammary gland-specific expression vector pEBS was constructed, which comprised of TGEV S gene driven by CSN2 regulator sequence and EGFP reporter gene.The mammary-specific expression vector pEBS was introduced into the mouse mammary cells (EMT6) using liposome. After 24-48 h, the expression of report gene EGFP was observed under fluoroscope. Then the transfected EMT6 were selected by G418 for two weeks. The positive cells were identifyied by PCR and RT-PCR, then induced by insulin+hydrocortisone+prolactin to express S protein. The recombinant S protein from culture supernatant of transgenic EMT6 was harvested after 48 h, The results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the transfected cells could expressed recombinant S protein, whose MW was 175 ku and the expression level was 2μg/mL.The recombinant S protein immunized BALB/c mice. The antibody titer in BALB/c mice following immunization with recombinant S protein was detectable after the first immunization. A titer of 7.28±0.15 ng/ml of serum of recombinant S protein specific antibody had been attained after the first boost which continued to increase to a level of 8.86±0.19 ng/ml after the second boost. In conclusion, the recombinant S protein expressed in mammary cells was able to elicit substantial immunological response against TGEV. This lays the basis for using mammary gland bioreactor generating edible vaccine.The vector pEBS mixed with liposome were injected directly into the mammary glands of the mice in later gestational period through the papillary ducts to transfect the glandular cells in vivo. The milk of the mouse in lactation period was collected on 1, 5, 10, 15 d after parturition. The results of ELISA assays showed that the maximum expression level of recombinant S protein in the milk was 3.50μg/L.The present study examined if injection of DNA into the testis could generate transgenic mice via seminiferous tubules injection. The mammary gland expression vector pEBS was mixed in proper ratio with liposome and trypan blue, then injected into seminiferous tubules of the mice testis. Five mice testis were injected with the transfected liquor, two mice died after surgery, three live males were used to mate with two ruttish females 5 days later. Twenty-six pups were produced and 8 of which were proven to be gene integration positive by PCR detection. Two were further confirmed to carry the S gene by Southern blotting analysis. It is concluded that transfecting spermatogonia in vivo can produce transgenic animals.