Generation Of A Monoclonal Antibody Against Transmissible Gastroenteritis Virus M Protein And Identification Of M Protein Epitopes

Transmissible gastroenteritis is a highly infectious disease of swine with symptoms ofdiarrhea, vomiting, and dehydration. TGEV is an enveloped virus with a positive-stranded RNAgenome, encoding four structural proteins. So far, epitope of spike protein (S) had been studieddeeply, however, that of membrane protein (M) were still unknown.In this research, polymerase chain reaction was performed for amplifying M gene withoutsignal peptides. Subsequently, truncated M gene was inserted into pGEX-6p-1vector, aprokaryotic expression vector, and recombinant M protein was expressed at the condition of “37℃,0.5mM IPTG and induction for6h”. The purified recombinant M protein was the foundation foranti-M protein monoclonal antibody (anti-M mAb) generation.According to the classic methods of generating mAbs, ultracentrifugated TransmissibleGastroenteritis Virus (TGEV) particles were used as immunogen and only one positive hybridomacell line secreting anti-M mAb was obtained and named as7G7finally. The results of indirectimmunofluorescence showed that anti-M mAb can recognize natural TGEV particles; the Westernblot results suggested that the denatured recombinant M protein also can reacted with anti-M mAb.It was revealed by indirect ELISA that the primarily analyzed epitopes located out of the virusmembrane. Moreover, the supernatant of7G7cells showed high specificity to TGEV, not to anyother kind of porcine virus. It was demonstrated that anti-M mAb would be a potential target for Mprotein epitopes identification.Ascitic fluid containing anti-M mAb were generated and purified and subsequently used astarget for phage display. After4modified rounds of biopanning,10random phages were amplifiedfor titer determination and affinity analysis. Compared to the phage library, which worked asnegative control, the whole10phages showed high affinity with the target. The sequencing resultsindicated that the10random shared the same12-amino acid (12aa) sequence, VHIDPHHQHDQM,which was named as phage-V. The alignment between exogenous12aa and M protein suggestedthat there were some similarity in the region of197aa to210aa of M protein and there were threekey points sharing the same amino acids sequences, which were199isoleucine (I),200asparticacid (D) and203proline (P). Finally, it was demonstrated by the competitive inhibition analysis that phage-V can block the recognition between either TGEV or recombinant M protein and theinhibition ratio was positively related to the phage-V inputs. In general, the exogenous12aa,which was identified by phage display, can either react with anti-M mAb or prevent the interactionbetween anti-M mAb to TGEV or recombinant M protein, suggesting function of exogenous12aa,for M protein epitope simulation. Because there were only three adjacent amino acids consistentto the M protein, it was inferred that exogenous12aa may be the spatial confirmation epitope of Mprotein and the motif may play crucial immunological roles.Conformational epitope of TGEV M protein was identified with the combination ofmonoclonal antibody construction and phage display, which would fill the gaps of related field andprovide the theoretical evidences for diagnosisi methods based on epitope and epitope vaccinedevelopment.