DNA Molecular Authentication And Protein Analysis Of Leech

Leech,a well-known animal-derived Chinese medicine,is the dry products of the whole body of Whitrmania pigra Whitman,Hirudo nipponica Whitman or Whitmania acranulata Whitman.Medically,it has been widely used as an anti-coagulation and anti-thrombosis agent in clinics for a very long time.In the Chinese herbal medicine market,the main source of leech is Whitmania pigra Whitman,and a small amount of Hirudo nipponica Whitman,and Whitmania acranulata Whitman is almost nonexistent However,the abundance requirement and high price of leech in market has caused serious intentional fraudulent modification of this medicine,and the similar morphological characteristics in different species of closer phylogenetic relationship and lack of professional experience can usually lead to unintentionally interfusing fake products.For instance,Poecilobdella manillensis Lesson was misused as medicinal leech quite often,and study had shown that it has different effects on thrombin and coagulation pathways with medicinal leech Whitmania pigra.Conventional methods for leech identification are based on the morphological characteristics of the intact adult leech which is not applicable for the larval and incomplete individuals yet.In addition,the Whitmania pigra Whitman as a major source of medicinal leech on the market,which the components that play the role of anticoagulant activity are still not clearIn this study,a reliable PCR-based approach was established for the identification of leech species,the newly developed method can be used for specific and rapid authentication of leech products for their form of raw material,the processed,and even the highly-processed.In addition,using SDS-PAGE and 2-DE electrophoresis for protein analysis of different leeches,confirm some protein bands from SDS-PAGE and protein spots from 2-DE which may provide a reference for leechs’ identification What’s more,we get an electrophoretically pure active polyprotein from Whitmania pigra Whitman,which identified as hypothetical protein from Comamonas testosteroni and has a molecular weight of 9985 through MALDI-TOF-MS analysisThe main research contents are as follows(1)Firstly,three pairs of specific primers were designed based on the difference of COI sequence between Whitmania pigra Whitman(W.pigra),Hirudo nipponica Whitman(W.acranulata),Whitmania acranulata Whitman(H.nipponica)and Poecilobdella manillensis Lesson(P.manillensis).The annealing temperature of the primers was optimized to determine the optimum PCR amplification reaction procedure Then,the specificity and applicability of the primers were verified.Finally,the primers were applied to the commercial products.In our study,W.pigra and W.acranulata shared one primer set and the length of its target product is 63 bp.Two pair of primers were selected for H.nipponica and P.manillensis respectively,and the expected amplicons for each species were 102 bp and 75 bp.The results of specificity and sensitivity test showed that all three pairs of primers had strong specificity and good sensitivity.The selected primers were applied to commercial products including raw,processed and highly processed leech,twelve batches of commercial samples were authenticated as medicinal leech products,while another six batches were counterfeited by fake leech,and one batch was adulterated products(2)Protein from different species of leech was extracted by ultrasound,then using SDS-PAGE and 2-DE gel electrophoresis to analyze protein,the gels were stained with coomassie brilliant blue.In the SDS-PAGE electrophoretogram of animal source of leech,there are 10 stable protein bands of W.pigra,9 stable protein bands of the H.nipponica,and the P.manillensis have 13 stable protein bands.According to the 2-DE result,the proteins in W.piga were mainly distributed below 45 kD,and there are 25 stable protein spots;the distribution of protein in the H.nipponica is concentrated,mainly in the pH 3-8 and molecular weight less than 66.2 kD,the alkaline side also has a stable of protein;the protein in P.manillensis is mainly distributed in three regions,respectively,I area is located in the acidic side and the molecular weight less than 25 kD,Ⅱ area is in neutral and the molecular weight less than 45 kD,Ⅲ area near the alkaline side and the molecular weight is less than 14.4 kD,in addition,there is also one point in alkaline that molecular weight about 25 kD.The difference of the distribution of protein spots may provide a reference for leechs’ identification.For raw and processed products,although the protein had degraded,they can be authenticated on the basis of the SDS-PAGE and 2-DE electrophoretogram.But for highly-processed products,most of the protein in the sample was degraded,and the quantity of protein bands(spots)decreased significantly,so speculated that processing technology had a great influence on the protein molecules of leech,it is difficult to identify processed products of leech according to its SDS-PAGE and 2-DE electrophoretogram.(3)Proteins from different species of leech were separated by SDS-PAGE,after in-gel tryptic digestion,then followed by MALDI-TOF-MS analysis.As the result shown,three proteins with high reliability were identified from W.pigra,WIV(39 kD)was identified as heat-inducible transcriptional repressor HrcA from Rhizobium sp.R339;WV(28 kD)was identified as DNA-binding response regulator from Bacillus sp.FJAT-26390 and WVIII(16.4 kD)was identified as glyceraldehyde-3-phosphate dehydrogenase from Hassallia byssoidea VB512170.Only one protein with high reliability from H.nipponica,and it was identified as predicted protein from Nematostella vectensis.In addition,there were two proteins with high reliability from P.manillensis,PII(47 kD)was identified as serum albumin from Bubalus bubalis and PV(20 kD)was identified as NAD-glutamate dehydrogenase from Paraburkholderia soli.(4)The anti-coagulant active components of W.pigra were extracted by refluxing with water after degrease with petroleum ether,the PRT result showed that the crude extracts had good anti-coagulant activity.The crude extracts were separated and purified by DEAE-52,Sephadex G-75 and Sephadex LH-20,getting an electrophoretically pure active polypeptide,and peptide were identified by MALDI-TOF-MS after in-gel tryptic digestion,result show that it is hypothetical protein from Comamonas testosteroni.