Research of Recombinant Influenza A Virus as a Vector for Mycoplasma
Pneumoniae P1a and P30a
Abstract
Methods: In this study, the major antigen genes P1a of MP adhesion
factor P1(3862-4554 bases) and P30a of P30(49-822 bases) were inserted
into the nonstructural protein (NS) gene of Influenza A virus strain
A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant
vectors NS-P1a or NS-P30a. The recombinant pHW2000 plasmids containing
NS-P1a or NS-P30a were co-transfected with the rest 7 fragments of PR8
into HEK293T cells. After inoculating chicken embryos, the recombinant
influenza viruses rFLU-P1a and rFLU-P30a were rescued. RT-PCR and
sequencing were used to identify the recombinant viruses. The
hemagglutination titers of rFLU-P1a and rFLU-P30a were determined after
5 successive generations in chicken embryos so as to indicate the
genetic stability of the recombinant viruses. The morphology of
recombinant influenza viruses was observed under electron microscopy.
Results: P1a or P30a was designed to be inserted into the modified NS
gene sequence separately and synthesized successfully. RT-PCR
identification of the recombinant viruses rFLU-P1a and rFLU-P30a showed
that P1a (693bp), P30a (774bp), NS-P1a (1992bp) and NS-P30a (2073bp)
bands were found, and the sequencing results were correct. After 5
successive generations, each virus generation has a certain
hemagglutination titer(from 1:32 to 1:64), and the band of P1a or P30a
can be seen in the corresponding positions. The virus particles under
the electron microscope appeared as spheres or long strips connected by
several particles, revealing a complete viral membrane structure
composed of virus lipid bilayer, hemagglutinin, neuraminidase, and
matrix proteins. Conclusion: The recombinant viruses rFLU-P1a and
rFLU-P30a which carried the advantaged immune regions of the P1 and P30
genes in MP were successfully constructed and identified. The genetic
stability of rFLU-P1a or rFLU-P30a was relatively high. The typical
morphology of influenza virus was observed under the electron
microscope. Our research provided a foundation for the further
development of MP vaccines for human.