The Mutations And Function Analysis Of Glycosylation Sites In HP-PRRSV Envelope Glycoprotein 5

PRRS was first reported in America in the 1980 s, then quickly spreaded all over the world. PRRS can cause porcine reproductive disorders, and respiratory symptoms, the disease is known as the "mysterious disease" at first, now renamed the porcine reproductive and respiratory syndrome. PRRS has brought a great threat to global pig industry.It is reported that some RNA virus evolutionary rate faster than others. Recently, in 2006 China broke out a "swine high fever", the disease caused by the main pathogen of the highly pathogenic porcine reproductive and respiratory syndrome virus(HP- PRRSV), clinical symptoms mainly for high morbidity and high mortality rate(20%). In order to more in-depth understanding of China’s epidemic situation, in 2006 after the outbreak of PRRS genetic evolution characteristics and highly pathogenic porcine reproductive and respiratory syndrome virus possible pathogenesis, we started the work.PRRSV GP5 is one of the variations of the highest protein, and one of the main structures of PRRSV capsule membrane glycoprotein, plays an important role in PRRSV infection, is essential for viral multiplication. GP5 protein has induced the body to produce neutralizing antibody linear table, is a good target protein in the development of the PRRSV new vaccines.Research data show that PRRSV in the process of natural infection, first to stimulate the body to produce the neutralizing antibodies(around 7 d after infection), and neutralizing antibodies produced by the time(about rehabilitation pigs and 28 d after infection) later. HP-PRRSV strains glycosylation sites are mainly exist in the GP5 protein. Study found PRRSV table is the main neutralizing antibody by glycosylation modified table B, the structural protein GP5 of N terminal functional areas(37-44 amino acid); GP5 another advantage of immune epitope, the neutralization epitope A, also in the N terminal(27-31 amino acids). Have the researchers speculate: table A interferes with the immune system aimed at neutralizing epitope B reaction, cause delays and neutralizing antibody titer.Research shows that in the middle of the table A and table B insert universal helper T cell epitope can enhance the immune effect of epitope B. Qi-sheng zheng will Cp G sequences and universal helper T lymphocytes table inserted between the GP5 table A and table B, and at the same time on two N34, N51 point mutations, glycosylation sites through the mice immune test proves that the constructed recombinant plasmid can stimulate immune mice in A relatively short period of time to produce higher levels of neutralizing antibodies.Porcine reproductive and respiratory syndrome(PRRS) caused huge economic losses to the global pig industry. In order to further reveal the HP- PRRSV capsule membrane glycoprotein 5 glycosylation amino acid mutation and function.This test is to contain the porcine reproductive and respiratory syndrome virus(PRRSV) SX- 1 strains of infectious c DNA cloning plasmid p VAX1- SX- 1 as a template, according to the principle of fixed point mutation, fusion by polymerase chain reaction(PCR) amplification GP5 protein 30, 34, 35, 44and51 asparagine glycosylation sites mutation sequence of the problem, replace the p VAX1- SX-1.Pieces of DNA plasmid encoding GP5 protein, respectively, to obtain recombinant plasmid p VAX1- N33 A, p VAX1- N34 A, p VAX1- N35 A, p VAX1- N44 A, p VAX1- N51 A. Transfection and the cultivation of 293 T cells respectively, 12, 24, 36, 48 hours after transfection cell sample collection, extract the RNA and reverse transcriptase to c DNA. And then by the method of RT-PCR amplification save virus GP5 protein coding sequence, sequence analysis showed that build correct. RT-PCR results showed that the GP5 glycosylation sites mutations affecting PRRSV transcription level. At the same time, 18, 36 hours after transfection cell samples collected using flow cytometry analysis, further understand whether PRRSV GP5 protein induced apoptosis, PRRSV GP5 protein glycosylation sites different mutant strains obtained will be in order to further reveal the GP5 protein glycosylation amino acid mutation function changes such as cell proliferation, cell cycle progression, apoptosis and viral replication, lay the foundation.Therefore, this test for different HP- PRRSV isolates GP5 protein glycosylation of different levels of transcription, the influence of its protein expression, and cause the body to produce a protective neutralizing antibody levels also vary, and participate in PRRSV and of the interaction between the host immune studies.