| Classical swine fever(CSF) is a highly contagious and fatal disease caused by swine fever virus(CSFV), which brings serious harm to the world pig industry. Recent years, with the depth of CSFV study,the development of new vaccines and the emergence of advanced diagnostic methods has played a significant role for the control of classical swine fever. NS2-3 proteins are nonstructural proteins of CSFV,which with good immune advantage.This study carried attenuated classical swine fever(Hog Cholera Lapinized Virus, HCLV)full-length genomic c DNA plasmid amplification epitope mass region of the gene of NS2-3, named as NS2-32, which length of 789 bp. The fragment and expression vectors p PICZαC were treated with Eco R I and Xba I double digested, and then connect to transform E.coli to obtain recombinant plasmid p PICZαC-NS2-32. After PCR, restriction analysis and sequencing test, recombinant plasmid p PICZαC-NS2-32 was linearized by Sac I single enzyme and electrotransformated Pichia pastoris X-33.Positive strains were screened by highly resistant, by changing the temperature, p H of the medium and methanol feeding. Inducing conditions include:(1) induced by temperature: 22℃, 24℃, 26℃, 28℃;(2)induction of p H value: 4.0, 5.0, 6.0, 7.0;(3) methanol concentration: 0.4%, 0.7%. Expression products by SDS-PAGE identification suggested that they were not the target protein. CSFV NS2-3 antigen concentration area expressing in a yeast expression system largely depends on the characteristics of the gene itself. Further analysis of the possible causes and influence factors of the target protein did not express,including: A and T content, excessive glycosylation, methanol concentration, protein degradation and impact of signal peptide etc. Further to obtain high expression, good antigenic protein and construction of CSFV antigen detection methods and the development of new vaccines which based on NS2-3 clusters to lay the foundation. |
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