Secretory Expression of Canine parvovirus Nonstructural Protein 1 (NS1) in Eukaryotic Cells Mediated by Human (Homo sapiens) CD5 Signal Peptide
WANG Xing1,2, LI Nan2, HUO Shan-Shan2,3, ZHANG Jian-Lou2,3, ZHANG Hui1,*, ZHONG Fei2,3,*
1 College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China; 2 College of Veterinary Medicine/College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; 3 Hebei Engineering and Technology Research Center of Veterinary Biotechnology, Baoding 071001, China
Abstract：Canine parvovirus (CPV) non-structural protein 1 (NS1) is an important functional protein involved in CPV replication and transcription. NS1 is also a pathogenic protein to the host. In order to explore the biological functions of NS1, recombinant NS1 proteins are needed. To prepare high-purity NS1 protein, the human (Homo sapiens) CD5 signal peptide sequence was used to mediate NS1 secretory expression in eukaryotic cells. First, based on human CD5 signal peptide sequence (GenBank No. NM_014207.4), a pair of complementary full-length CD5 signal peptide sequences (2 oligos) with restriction sites at both ends were designed and synthesized. The CD5 signal peptide gene was then produced by annealing the 2 oligos. The resulting CD5 signaling peptide gene was inserted into a NS1 eukaryotic expression vector pcDNA3.1-NS1/H at the 5' end of the NS1 gene by the restriction sites and fused with the NS1 gene to generate the NS1 secretory eukaryotic expression vector pcDNA3.1-CD5-NS1/H. The NS1 vector was transfected into human embryonic kidney (HEK) 293T cells, and the expressed NS1 protein was purified by affinity chromatography with Ni-NTA agarose beads from the cell culture medium. The detection results of SDS-PAGE and Western blot showed that the high-purity NS1 protein was obtained (>95%) and it could specifically react with anti-His antibody, indicating that the constructed NS1 vector could mediate NS1 secretory expression in eukaryotic cells. In order to achieve a high level of expression, the effects of the vector doses, transfection time and expression time on NS1 expressions were analyzed and optimal conditions for NS1 secretory expression were preliminarily determined as vector dosage 30 μg in T75 cell culture flask, transfection time 5 h, expression time 48 h. This study could provide basic materials for further investigation on NS1 biological functions.
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