In order to express and purify Giardia duodenalis cyst wall proteins CWP2 and CWP3 in vitro and analyze their bioinformatic characteristics,this study amplified the CWP2 and CWP3 genes from the whole genomic DNA of G.duodenalis by PCR.The expression plasmids pET-28a-CWP2 and pET-28a-CWP3 were constructed and transformed into E.coli BL21(DE3)competent cells after enzyme digestion and sequencing identification.After IPTG induction and ultrasonication,the protein was purified by urea and detected by SDS-PAGE and western blot.The SDS-PAGE results showed that the recombinant CWP2 and CWP3(rCWP2 and rCWP3)were 38.79ku and 27.38ku,respectively,and the two recombinant proteins were mainly expressed in the form of inclusion bodies.The purified protein bands were single and the purity was more than 90％.Western blot results showed that the two recombinant proteins had good reactogenicity with His-tag MAb.Measued by BCA method,the concentrations of rCWP2 and rCWP3 were 0.207mg/mL and 0.465mg/mL respectively.The bioinformatics properties of these two recombinant proteins were predicted by bioinformatics software such as Prot Param,Prot Scale,Singal P 6.0,Deep TMHMM,Net Phos 3.1,SOPMA,SWISS MODEL and STRING.The results showed that G.duodenalis CWP2 and CWP3 were unstable and stable proteins,respectively;each of them had a signal peptide,but no transmembrane region;CWP2 and CWP3 had 39 and 25 phosphorylation sites respectively,and all formed oxidative(O)phosphorylation;the main secondary structures of CWP2 and CWP3 were α-folded and irregularly coiled;the predicted tertiary structure model of CWP2 showed 100％homology to E2RTS7.1.A of G.duodenalis WB clones C6 strain;the predicted tertiary structure model of CWP3 showed 90.69％homology to E1F7Q8.1.A of G.duodenalis WB clone P15 strains;the tertiary structure model also showed that the two proteins were mainly α-helix and random coil,which was consistent with the secondary structure;the STRING software predicted that the CWP2 interacts with seven proteins,including α-1 giardin,DNA helicase,and CWP3 interacts with α-2 giardin,δ-giardin and β-giardin.The above results showed that high purity rCWP2 and rCWP3 were obtained in this study,and both had good reactogenicty,the main bioinformatics characteristics of the two were analyzed,which lays the foundation for further exploration of cystal wall proteins in G.duodenalis.
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