Objective To clone human cell division cycle gene 2(CDC2)from human liver cancer tissue and express CDC2 protein in E.coli BL-21(DE3).Methods The total RNA was extracted from liver cancer tissue and amplified by reverse transcription polymerase chain reaction(RT-PCR).The PCR products were cloned into the prokaryotic expression vector pET28a(+)followed by DNA sequencing.The recombinant pET28a(+)/CDC2(rCDC2)plasmid was transformed into E.coli.The rCDC2 was induced with IPTG and characterized by SDS-PAGE.Results The cloned CDC2 gene was composed of 894 nucleotides,and is accordance with that reported in GenBank.The prokaryotic expression vector was constructed successfully.The CDC2 protein was successfully expressed in E.coli.Conclusion The CDC2 cDNA is successfully cloned from human liver cancer tissue,and can express in E.coli.

Objective To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. Methods The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. Results SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. Conclusions The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.

Objective To clone the rat heme oxygenase-1(RHO-1) from rat spleen and express RHO-1 in E.coli BL-21.Methods The total RNA was extracted from rat spleen and amplified by reverse transcription polymerase chain reaction(RT-PCR).PCR products were cloned into pMD18-T(TA)vector followed by DNA sequencing.RHO-1 cDNA fragments in TA vector were subcloned into the prokaryotic expression vector pET28a(+).The recombinant pET28a(+)/RHO-1(rRHO-1) plasmid was transformed into E.coli.The rRHO-1 was induced with IPTG and characterized by SDS-PAGE.Results The cloned RHO-1 gene was composed of 870 nucleotides,and was accordance with the sequence reported in GenBank.The prokaryotic expression vector was constructed successfully.The RHO-1 protein was successfully expressed in E.coli.Conclusion The prokaryotic expression vector of rRHO-1 has been constructed,and the fusion protein has been successfully expressed.