Prokaryotic expression and polyclonal antibody preparation of peste des petits ruminants virus H protein

Abstract:

[Background] Peste des petits ruminants (PPR) is an acute, severe and contagious infectious disease caused by peste des petits ruminants virus (PPRV), which is endangering the sheep/goat-farming in Asian and African countries. [Objective] The objective of this study is to express PPRV H protein prokaryotically and to prepare its polyclonal antibodies. [Methods] According to the sequence of PPRV Tibet strain deposited in GenBank, h gene was optimized based on the codon usage bias of E. coli and synthesized by using two-step PCR. Then, the sequencing confirmed h gene was inserted into prokaryotic expression vectors pET-28a, pET-30a and pET-32a, respectively. The constructed recombinant plasmids pET-28a(-30a and -32a)-H were then transformed into E. coli BL21(DE3) for expression under the induction of IPTG. The expressed H protein from E. coli BL21(pET-30a-H) were purified by cutting out the aimed band from the SDS-PAGE gel. Polyclonal antibodies were prepared by immunizing New Zealand white rabbits with the purified H protein. [Results] The recombinant H protein expressed by E. coli (pET-28a (-30a, -32a)-H), with a relative molecular mass of about 70, 68 and 86 kD, respectively, mainly existed in a form of inclusion body, of which the expression level reached the maximum at 7 h after induction. The polyclonal antibodies prepared by immunizing the rabbits with the purified H protein from E. coli BL21(pET-30a-H) showed specific reactivity with expressed recombinant H protein. The titer values of the serum ranged between 1:6 400 to 1:25 600. [Conclusion] PPRV H protein was successfully expressed in prokaryotic cells, and high titer anti-H protein polyclonal antibodies were obtained, which laid a foundation for further research on the function of H protein, and the fine linear B cell epitope mapping of PPRV H protein.