Flounder Mx Gene Cloning, Sequence Analysis And Expression In Pichia Pastoris

Mx protein, one of the antiviral proteins induced by type I interferons (IFN),can induce the antivirus state of the cells and thus has a broad-spectrumantivirus effects. Mx proteins has been identified in mammals including human,avains and some kinds of fishes. Much attention has been put to the Mxproteins ever science 1990s. In this paper, cDNA sequence of Southernflounder Mx gene was isolated by the methods of reverse transcriptionalpolymerase chain reaction (RT-PCR). Then a pPIC9K-Mx expression vectorwas constructed and transformed into Pichia GS115 strain. In addition, the Mxprotein was successfully expressed in the yeast strain under the induction ofmethanol. The results will provide some important and basal data for thefurther studies on the antivirus mechnisms of fish Mx proteins.First total RNA was extracted from the kidney of Southern flounder withTriZol agent and cDNAs were subsquently sythesized by reverse transcription.We searched for the DNA and protein sequences of Mx protein familymembers ever reported within the website of the GenBank of NCBI. Then Allthe sequences were edited and megaligned by the Clustal methods in theDNASTAR software package. Primers for amplificating the cDNA sequence ofSouthern flounder Mx gene were then designed according to the results ofaligning. RT-PCR was then performed and a cDNA fragment obtained asexpected. The purified RT-PCR product was ligated into pEM-T vector andtransformed into E.coli DH5a strain for sequencing. Sequence analysis resultsshowed that the fragment amplified was 1980 bps in length with an ORF of1890bps which coded for 629 amino acids. The deduced molecular weight ofthe predicted protein was about 71.7kDa. The deduced amino acid sequencewas submitted and blasted and aligned with the homologs deposited inGenBank. Results showed that the deduced Southern flounder Mx proteincontained all three features that are highly conserved in all IFN-induced Mxproteins of vertebrates, that is a triparitite guanosine-5' -triphosphate(GTP)-binding motif (GXXXSGKS/T, DXXG, T/NKXD), a signature of the dynaminfamily (LPRGS/KGIVTR), and a highly conserved leucine zipper motif in theC-terminal region of the proteins. Thus the full-length cDNA sequence ofSouthern flounder was isolated in our experiments. Megalign results showedthat the percent of amino acid identities between Southern flounder Mx proteinand other family members varied from 75.1% to 98.7% in fishes and 50.3% to55.1% in mammals.To express the Mx protein of Southern flouder in pichia, a shuttle vectorpPIC9K was chosen, which can express fusion protein both in pichia and E.coli. After transformation in E. coli, the constructed plasmid pPIC9K-Mx wasextracted and linearized with the restrict enzyme sacI, then transformed intopichia via electroporation under 750V/4.3ms conditions. The transformed clonewas plated on MGY medium,and the property of GS115 enabled only thetransformed one could grow on the selective medium. The integrative state ofMx cDNA within the Pichia genome was also confirmed by PCR amplificationand subsequent sequencing. The results of SDS-PAGE showed that SouthernMx protein was successfully expressed in Pichia under methanol induction andthen secreted into cultured supernatant.