Prokaryotic Expression of Affinity Peptides to Membrane Protein of Transmissible gastroenteritis virus and Analysis of Viral Affinity Activity

doi:10.7668/hbnxb.201751554

Abstract

Abstract: In order to investigate the viral affinity of Transmissible gastroenteritis virus of swine (TGEV) membrane protein (M protein) affinity peptide and establish a rapid and effective TGEV detection method, a tandem gene containing the TGEV M protein affinity peptide was designed. The nucleotide sequence was optimized according to the E. coli preference codon. A recombinant plasmid pUC57-MQHT containing M protein affinity peptide gene was obtained by artificial gene synthesis. According to the sequence of the synthetic gene, a specific primer was designed and synthesized. And then, using pUC57-MQHT as a template, TGEV M protein affinity peptide gene (about 150 bp) was obtained by PCR. The affinity peptide gene was respectively inserted into the Bam H Ⅰ/Xho Ⅰ multiple cloning sites of the prokaryotic expression vectors pET-32a and pGEX-6p-1 to obtain the recombinant plasmids pET-32a-MQHT and pGEX-6p-MQHT. The recombinant plasmids were identified by single enzyme digestion of Bam HⅠ, double enzyme digestion of Bam HⅠ/Xho Ⅰ and sequencing. The recombinant plasmids were transformed into E. coli Rosetta (DE3) and induced by IPTG to obtain the expression of recombinant proteins TRX-MQHT and GST-MQHT, respectively. The molecular weights of the two recombinant proteins were 25, 31 ku, respectively. After purification, the recombinant proteins TRX-MQHT and GST-MQHT were identified by His-tag monoclonal antibody and GST-tag monoclonal antibody, respectively. The results indicated that the purified proteins obtained were the target proteins. Western Blot analysis showed that the two recombinant proteins could specifically bind to TGEV. Dot-ELISA analysis indicated that the two recombinant proteins showed positive reaction to TGEV at 1×10³, 5×10² and 2.5×10² TCID50/mL, but no visible spot was found at 1.25×10² TCID50/mL. The results showed that the two recombinant proteins had good affinity to TGEV virions. Dot-ELISA analysis showed that the minimum binding titer of the two recombinant proteins to TGEV virions was 2.5×10² TCID50/mL. Blocking experiments showed that TGEV-positive serum diluted 1:100 or 1:200 could completely blocked the binding of the two recombinant proteins to TGEV H strain (1×10⁵ TCID50/mL). The results showed that the specificity of the two recombinant proteins was good. This study laid a certain theoretical and material basis for the establishment of TGEV diagnostic methods.

CLC Number:

S858.28

YU Tianfei, DONG Huiying, XIE Pengyu, SUN Wanshu, YIN Haichang, LI Ming, YU Zhidan. Prokaryotic Expression of Affinity Peptides to Membrane Protein of Transmissible gastroenteritis virus and Analysis of Viral Affinity Activity[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(5): 224-230. doi: 10.7668/hbnxb.201751554.

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