| Objective:Leech is a traditional Chinese medicine.The extraction and separation technology of anticoagulant constituents from Poecilobdella manillensis Lesson,which is widely distributed in Hainan,was studied in this paper.Combining with mass spectrometry identification and bioinformatics analysis of transcriptome,the experiment and theoretical basis were provided for the development of P.manillensis in Hainan.Methods:1、Different parts(head and whole),different extraction methods(acetone method,water extraction and alcohol precipitation method,salt extraction method,water extraction method)and ultrafiltration membranes(3k,10 k,30k)with different molecular weights of the anticoagulant activity on P.manillensis was compared by the method of thrombin titration.The difference between test tube titration and white plate titration was discussed.2 、 The anticoagulant components was extracted and separated by 0.9 % saline solution,membrane ultrafiltration,gel filtration and high performance liquid chromatography,The anticoagulant components were detected by LC-MS/MS and compared with protein Uniprot database.3、Two samples of P.manillensis and two samples of H.nipponica were selected to construct transcriptome sequencing library and sequenced by RNA-seq.All Unigenes obtained by sequencing were classified by GO,COG and KEGG.The results were compared with those obtained by mass spectrometry.Functional annotations and Pathway annotations were given to predict the coding region of Unigene protein.Results:1、Using thrombin tube titration,the anticoagulant activity of 0.9 % saline extract from the head of P.manillensis was the highest,reaching 1811.11 U/g,RSD 1.84%.The molecular weight of anticoagulant component was about 15.96 k D by selecting10 k D and 30 k D ultrafiltration membranes.2、The crude extract of P.manillensis was filtrate by Sephadex G-50 gel column and purified by C18 column of HPLC.Two active peaks were obtained.By LC-MS/MS,the activity peak 1 matched 5 proteins in the Uniprot protein database,and the activity peak 2 matched the 3 protein in the Uniprot protein database,and the protein scores were all low.3、39953256 of P.manillensis sequences were obtained,and 40650 Unigenes were assembled,with an average length of 987 nt and N50 length of 1948 nt.40239 948 sequences of H.nipponica were assembled to produce 40623 Unigenes with an average length of 957 nt and 1876 nt.According to those database prediction,an anticoagulant gene with 83 amino acid was obtained,and the similar ratio to HM1 was 81.707%.The active peak 1 matches two Unigenes in the predicted gene bank of the transcriptome of P.manillensis.Unigene in the transcriptome of P.manillensis can be roughly divided into 25 categories according to KOG function,33 branches according to GO function,including biological process,cell component and molecular function,and data in the transcriptome can be divided into 6 categories,34 subcategories and 384 KEGG pathways according to the metabolic pathway with KEGG database as reference.Unigene in H.nipponica transcriptome can be roughly divided into 25 categories according to KOG function,47 branches according to GO function,which can be divided into three categories: biological process,cell component and molecular function.With KEGG database as a reference,data in the transcriptome can be divided into six categories,33 subcategories and 379 KEGG pathways according to metabolic pathways.Conclusion:Two anticoagulant peaks were isolated from the head of P.manillensis,the activity peak 1 matched 5 proteins in the Uniprot database,while the activity peak 2 matched3 protein in the same database,and both protein scores were low.The high-throughput sequencing technology was used to sequence and analyze the transcriptome of the H.nipponica and P.manillensis,which showed that the two kinds of leeches have certain biological consistency.It provides abundant data resources for the development of P.manillensisand the further substitution research of leeches. |
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