Structure Interpretation Of PMetMb And Development Of Its Sandwich-ELISA For Meat Color

Meat color is an important meat quality for effect on consumers purchase.As all known,meat color is usually a special protein color from myocyte,called myoglobin(Mb).It’s necessary to develop a specific and accurate diagnostic method for meat color evalution in pork industry via analysis of Mb molecular information.In this research,purified porcine metmyoglobin(pMetMb)was exctrated from its myocardium,and its molecular information and structure characteristics were interpreted by mass spectrometry(MALDI TOF/TOF)with bio-information technology.As immunological knowledge,pMetMb could be to prepare its specific polyclonal antibody(PcAb)and monoclonal antibody(McAb)so that a method of double-antibody sandwich ELISA for meat color evalution would be built up.1 pMetMb molecular structure informationpMetMb appared brownish red color and 17kDa molecular weight.It was a single subunit protein with a polypeptide chain and a heme-Fe2+/3+group.Its peptide chain consisted of 133 amino acids(AAs)at least,in which contained 15 kinds of AAs.Comparation of AAs kind,number and rate between pMetMb and horse myoglobin(hMb),three of them were matched up to 90%between pMetMb and hMb.And then compared to pMetMb and Mb gi|494385 in NCBI database,5 peptide fragments in two proteins matched 79.9%(100%confidence,P<0.01).As these results,pMetMb compared with hMb or Mbgi[494385 were high homological protein.pMetMb was only tertiary structure.Its secondary structure contained 7 a-helix and 2 3 10-helix.Because of 3 10-helix,its spherical spatial structure folded tighter for electron transfer and color stability.Porphyrin ring(heme-Fe2+/3+)in pMetMb was a key functional group,located at 6th α-helix and 7th a-helix,N-bond on heme-Fe2+/3+usually bound with His64 and His93 in imidazole group as different chemical state.When heme-Fe2+/3+ was happened oxidation-reduction reactions continuously,the meat color was keeping ideal.Because pMetMb was a major and important color material in muscle cell,purified pMetMb could be a better antigen for fast immune-diagnosis of meat color.2 Prepared pMetMb-McAb and-PcAbpMetMb was injected into skin several pits on rabbits and BALB/c mice back.After 4 times injection,some specific anti-serum from rabbits was collected and tested its titer.Its titer was over 1 x 105 called as PcAb.After immune,some mice splenocytes were fused with SP2/0 cells to produce four hybridoma cell lines.One of them,as 4F2 cell was used to prepare mice ascite.Then this ascite was purified and tested its titer.When ascite titer was up to 1×105,this was purified to prepare McAb for study of meat color-sandwich ELISA.3 Meat color-sandwich ELISApMetMb was as antigen to establish a method of double-antibody sandwich ELISA for meat color test.McAb/bottom-pMetMb-PcAb/top was chosen as a better model.Under this model,the optimal conditions of sandwich ELISA were 4μg/mL McAb,1%casein/60min,pMetMb/90min,4μg/mL PcAb/90min,1:4000(V:V)2nd Ab/45min.Only McAb cultured under 4℃ overnight,others cultured under 37 ℃.pMetMb standard curve was built up.Arranging 0.04-5.120μg/mL pMetMb,its linear equation was Y=0.4959X-0.5788(R2=0.9919,P<0.01).This method baseline was 0.027p.g/mL pMetMb(±3SD),total recovery rate was 100.5%.Furthermore,13 pork samples were tested and their pMetMb recovery rate was 86.0%-87.3%.There was no difference between batches(P>0.05),while there was very significant difference among three pMetMb concentrations(P<0.01).These were indicated this model of sandwich ELISA was stability,very high accuracy and sensitivity.