Expressing Of Structural Proteins N And F From Human Metapneumovirus In E. Coli, Yeast And Insect Cells

Human metapneumovirus(hMPV) is a newly identified virus of the Paramyxoviridae family.It was first reported in June 2001 as a cause of acute respiratory tract disease in Dutch children(van den Hoogen,2001).This new virus displays a high percentage of sequence identity with avian pneumovirus(APV). hMPV is a negative-sense,nonsegmented single-strand RNA virus composed of eight genes,namely,nucleoprotein(N),phosphoprotein(P),matrix protein(M),fusion protein(F),second matrix protein(M2 and M2.2),small hydrophobic protein(SH), attachment glycoprotein(G),and RNA-dependent RNA polymerase(L) in the order of 3'-N-P-M-F-M2-SH-G-L-5'。hMPV has been detected as a cause of upper and lower respiratory tract diseases in both children and adults,especially in very young children,elderly persons,and immunocompromised patients.This virus may be responsible for 10%of acute respiratory tract infections(ARIs) which were negative for all other common respiratory viruses,such as respiratory syncytial viruses(RSVs),influenza viruses, parainfluenza viruses and adenoviruses.More and more scientists have been attracted by this newly identified virus.The virus is difficult to be detected by cell culture,due to its selective,slow growth and mild cell pathogenic effect,it is not easy to be diagnosed by conventional methods.Reverse transcriptase PCR(RT-PCR) is used currently for detecting of the virus in clinical samples and the need for a reliable and rapid diagnostic method is obvious.Producing the antigen by recombinant DNA technology for analyzing of hMPV is an accessed way to understand the characteristic of this newly discovered hMPV and the information about the prevalence of this virus in China. The genomes of the hMPVs detected from children in Beijing have been sequenced.The effort to isolate the virus from clinical specimens is under way.To obtain enough antigens of this virus,the structural proteins N and F from hMPV were expressed in three expression systems including E.Coli,yeast and insect cells.The N protein with the encoding gene of 1182 bp in length and predicted 394aa is a nucleoprotein which plays a role in encapsidation regulating the transcription and replication of the viral genome.It is a major immunogen with highly conserved amino acid sequence The F protein with the encoding gene of 1620 bp in length and predicted 539 aa is a major surface glycoprotein which promotes fusion of the hMPV and cell membranes.It was indicated to be a major antigenic determinant that mediated effective neutralization and protection against hMPV.At the first step,6 His-tags and several restriction sites were cloned in N terminus of N protein encoding gene for purification of protein easily to constructed recombinant pBh1887N(K) for Bac-N-blu system and recombinant pFh1887N for Bac-to-Bac system.The correctness of the recombinant DNA was determined by sequence analysis.There was blue plaque-forming at Bac-N-blu expression system and N gene can be amplified from recombinant virus of culture medium at Bac-to-Bac expression system,indicating that the N gene has been inserted in baculovirus genome.The lysate of sf9 insect cells at 72h after infected was subjected to SDS-PAGE and Western blot using antibodies against His and N protein of hMPV, respectively.The negative results indicated that N protein was not expressed as expected.It suggested that the His-tag located at the N-terminus of the inserted gene could interfere the expression of N protein,so the His-tag was transferred to the C-terminus of the gene by re-cloning of the gene and expressing this recombinant gene in several expression vectors as the second step.The recombinant pB1887N for Bac-N-blu vector,recombinant pF1887Nh for Bac-to-Bac vector,single or 2 copies recombinant pAOa1887Nh for yeast expression system and recombinant pET1887Nh for E coli were constructed and expressed in corresponding express system,respectively.Target protein was detected in the baculovirus-insect cell system 72h after the cells were infected by recombinant virus,as determined by SDS-PAG and Western blot.In yeast expression system,a band with a molecular weight of approximately 43kD was revealed when the culture supernatant were separated by SDS-PAGE,which was absent in culture supernatant for negative control,however there were not any target protein detected in Western blot although target N gene could be amplified from recombinant yeast genome.The N protein was expressed in BL21(DE3) successfully and could be detected by Western blot using antibodies either against His or N protein. Scale up and purification of N protein expressed by baculovirus-insect cell system for hMPV serological test are under way.Based on experiences of N protein expression,the recombinant pB1816F for Bac-N-blu vector and the recombinant pF1816Fh for Bac-to-Bac vector were constructed and the correctness of the recombinant DNA was determined by sequence analysis.The lysate of sf9 insect cells at 72h after infected in Bac-to-Bac system was subjected to SDS-PAGE and Western blot using antibodies against His and F protein of hMPV,respectively,a band with a molecular weight of approximately 60kD was observed by Western blot.There was blue plaque-forming at Bac-N-blu expression system which indicated that F gene has been inserted in baculovirus genome.The next experiments are to select and amplify F protein recombinant baclulovirus.In this study,hMPV structure proteins display different expression level in three expression systems.The N protein is easy to express in E coli,but F protein is not,probably because of its toxic effect to cells and the complex structure of the protein.The N protein maybe be modified when expressed in yeast,so can not be detected by Western blot because N-special antibody does not recognize modified protein.Both N and F protein can be expressed in the baculovirus-insect cell system, the expression quantity is low.It maybe due to low MOI and unhealthy insect cell during infection.Two recombinant N protein vectors were constructed.One has a N-terminus His-tag and the other a C-Terminus His-tag respectively.Only the N gene with C-Terminus His-tag got expression.It was considered that complex secondary structure of DNA or mRNA perhaps appears at N -terminus of the N gene with N-terminus His-tag,so these secondary structure inhibited protein expression.This is important to be considered in other protein expression experiments.We expect to find an ideal expression system to obtain large amount of hMPV proteins by investigating the expressional information of hMPV N and F protein in three expression systems.The protein purified from ideal expression system can be applied to develop serological diagnostic techniques and VLPs assembling in future.