Identification Of Antler Derived Chinese Medicines Based On Species-specific Primers Amplification And Discovery Of Signature Proteins

Cervus antler,antelope antler and other antler derived Chinese medicines（ACM）are the horns of specific animals and their processed products.Cervus antler is a tonic derived from the ossified antlers or exfoliated antler base of Cervus elaphus Linnaeus（CEL）or Cervus nippon Temminck（CNT）;while pilose antler is the unfossified and fuzzing antler of CEL or CNT;and antelope antler is the horn of Saiga tatarica Linnaeus（STL）.Their curative effect is exact and remarkable.Recently,in the market of Chinese medicine,there is a great demand for most ACM,such as pilose antler and antelope antler.However,their limited animal resources have promoted the emergence of substitutes or counterfeits for ACM,which may pose a threat to the safety and effectiveness of these medicines in clinic.At present,the identification of ACM mainly depends on its characteristics,which is highly subjective and requires professional literacy,and it is especially difficult to distinguish their morphological characteristics after raw materials are processed.Hence,it is not enough to reliably identify the authenticity and the animal origin of ACM by traditional identification methods.In this study,species-specific primers were designed and screened according to the differences of mitochondrial gene between original animals of Cervus antlers,antelope antlers and their counterfeits,and The PCR technique was established to identify the authenticity of Cervus antler and antelope antler based on species-specific primers amplification.In addition,SDS-PAGE and 2-DE electrophoresis were adopted to find and identify the differential proteins between Cervus antler and pilose antler,which could provide a basis for distinguishing them;then their SDS-PAGE protein bands were analyzed by MALDI-TOF/TOF-MS to identify signature proteins.The main research contents are as follows:（1）Firstly,candidate primers were designed and selected based on the differences of mitochondrial gene between CEL,CNT,reindeer as well as sambar by DNAMAN and Oligo software;and three sets of primer with good specificity were screened according to the brightness of the target bands by PCR amplification of DNA templates from Cervus antlers and their counterfeits.Then PCR conditions were optimized and the specificity as well as sensitivity were investigated,and the reference sample mixtures in different proportions were detected.Finally,the established method was used to identify the authenticity of commercial Cervus antlers.The results showed that three primer sets designed and screened in this study had high specificity,in which Cervus elaphus and Cervus nippon antlers shared the same primer set,and the length of target amplicon was 187bp,meanwhile,the amplicons length of reindeer and sambar antler were 200 bp and 101 bp,respectively.Under the optimized PCR amplification conditions,the specificity was high and there was no cross-reaction between primers.The detection limit of primer PRT was 1 ng·μL^-1,while that of PC and PRU was 10 ng·μL^-1.This technique can detect CEL,CNT,reindeer as well as sambar in reference sample mixtures,indicating that the established method can be used to identify adulterated products of Cervus antlers.The detection of 14 batches of commercial Cervus antlers showed that 9 batches were authentic and the other 5 batches were fake reindeer antlers.（2）Candidate primers were designed and selected based on the differences of mitochondrial gene between STL,Ovis aries Linnaeus（OAL）,Procapra gutturosa Pallas（PGP）and Capra hircus Linnaeus（CHL）by DNAMAN and Oligo software;and four primer sets were screened by PCR amplification of DNA templates from antelope antlers and their counterfeits.Then simplex PCR conditions and multiplex PCR system were optimized,and specificity as well as sensitivity were investigated,then the reference sample mixtures of antelope antlers and their counterfeits in different proportions were detected.Finally,the established method was used to identify the authenticity of commercial antelope antlers.The results showed that four primer sets designed and screened in this study had high specificity without cross-reaction under the optimized simplex PCR.Meanwhile,the amplicons showed clear bands and no non-specific amplification after PCR of mixed DNA template in the optimized multiplex PCR system and the detection limit was 10 ng·μL^-1.This technique can detect STL,OAL,PGP and CHL in reference sample mixtures,indicating that the established method can be used to identify adulterated products of antelope antlers.The detection of 12 batches of processed antelope antlers showed that 7 batches were authentic,and 2 batches were adulterated products containing Ovis aries antler and Capra hircus antler,and the other 3 batches were fake products from Ovis aries antler and Capra hircus antler.At the same time,3 batches of antelope antler TCM were detected,and there was not band in the electrophoretic results.（3）The proteins of Cervus antler and pilose antler were respectively extracted by homogenized and ultrasonic method to be analyzed by SDS-PAGE and 2-DE electrophoresis.The SDS-PAGE results showed that there were two clear protein bands from Cervus antler and four bands from pilose antler,and these stable protein bands can be used as basis to distinguish them.In addition,3 batches of pilose antler and 5 batches of Cervus antler slices were analyzed by SDS-PAGE.It was found that2 batches of pilose antlers had the characteristics of protein bands of Cervus antler,which were identified as fake pilose antlers,and the other batches were conformed to the corresponding protein characteristics of Cervus antler and pilose antler.The 2-DE results showed that the protein spots of Cervus antler were mainly in I area with pH5⁷ and molecular weight at ca.45 kD,and II area in pH 10 and molecular weight at ca.66.2 kD.While the spots distribution of pilose antlers were concentrated in pH5⁷ and molecular weight at ca.45 kD（III area）,and there were also three repetitive protein spots in pH 5⁶ and molecular weight at ca.20 kD.There were differences in2-DE profiles,which can provide further reference for the identification of Cervus antler and pilose antler.（4）The SDS-PAGE protein bands of Cervus antler and pilose antler were selected to develop in-gel tryptic digestion,MALDI-TOF/TOF-MS analysis and database matching.The identification results of protein bands from Cervus antler were all less than 80 points,indicating there was not reliable protein.While three proteins with high reliability were identified from protein bands of pilose antler.Band II and III were respectively identified as albumin（ALB）and hypothetical protein from Cervus elaphus hippelaphus,which could be reference for identification for Cervus antler and pilose antler.While band I was identified as hypothetical protein from Streptomyces...