Preparation,Identification And Application Of Sericin Antibody Of Silkworm

Bombyx mori is not only a representative insect of lepidoptera,but also a silk secreting insect with important economic value.The silk gland is the only silk secretion organ of silkworm,which can efficiently synthesize and secrete silk proteins,silk protein including silk fibroin and sericin.Silk fibroin protein is synthesized and secreted by posterior silk gland(PSG),sericin is synthesized and secreted by middle silk gland(MSG).Sericin mainly includes Sericin 1(Ser1),Sericin 2(Ser2)and Sericin 3(Ser3).The silk of silkworm is mainly composed of two sericin fibers wrapped with fibroin.Silk protein,as one of the most important and valuable research hotspots of silkworm,has been paid much attention.With the development of science and technology,silk protein related materials have been used in many scientific fields.In the past,due to the lack of Sericin antibody,the detection of sericin expression at the protein level and the application of sericin antibody detection have not been reported.Therefore,by preparing sericin antibody of silkworm and applying it to basic experiment and application research,this experiment gives full play to the potential application value related to sericin antibody,and mainly obtains the following research results:1.Several different sericin antibodies were successfully preparedIn this experiment,Ser-1B,Ser2-2 and Ser3-2 amino acid sequences of sericin genes of silkworm were firstly retrieved from the NCBI database.According to the antibody design software,seven polypeptides were selected for synthesis and prepare antibodies.The antibody titer was detected by Elisa test,and all antibody titers were over 1:80000.Six sericin antibodies with good specificity were screened as Ser1-1,Ser1-2,Ser2-1,Ser2-3,Ser3-1 and Ser3-2 after specific detection of antibodies with synthetic peptides.2.Identification of silkworm sericin antibody specificitySilkworm sericin protein expressed in different parts of the middle silk gland(MSG)of the silkworm,for specific identification of the sericin protein antibody,first to V5^thD larvae of silkworm 872 strains of the anterior segment of middle silk gland(A-MSG),the middle segment of middle silk gland(M-MSG)and the posterior segment of middle silk gland(P-MSG)contents in the immune hybrid(Western blot)experiments.Results showed Ser1 antibodies in the 872 M-MSG and P-MSG respectively detected two immune stripe,detected in A-MSG not immune stripe,the results show that in the period of Ser1 only expressed in M-MSG and P-MSG,it with the current Ser1 gene in M-MSG and P-MSG expression results are similar.The antibody of Ser2 was detected in A-MSG with two immune bands,and no signal was detected in M-MSG and P-MSG.Ser3 antibody detected the signal in A-MSG and M-MSG,but not in P-MSG.The above results were consistent with the reports of sericin gene at nucleic acid level.Subsequently,immunohistochemical experiments were carried out on the middle silk glands of silkworm 872 strain of the V7^thD larvae.The results showed that Ser1 antibody detected fluorescence signals in sericin layer of A-MSG,sericin layer and cell layer of M-MSG and P-MSG.Ser2 antibody detected fluorescence signal only in sericin layer and cell layer of A-MSG.Ser3 antibody detected signal in sericin layer of A-MSG and cell layer of M-MSG,but not in P-MSG.The above results were consistent with the results of western blot.Finally,we identified these sericin antibodies,indicating that they can be used in subsequent application experiments.3.Sericin antibody was used to observe the secretion process of silk proteinThe dynamic secretory process of sericin was observed by using the specific sericin antibody.Paraffin sections of A-MSG,M-MSG and P-MSG of the 7th day larvae of the 5th instar of the 872 strain of silkworm were carried out.After HE staining,it was found that the distribution of sericin layer of A-MSG was rich,so I,II and III were selected for detailed observation.The distribution of sericin in the sericin layer was studied by immunohistochemistry.Only a small amount of signal was detected in sericin layer of area II by Ser1 antibody.The signal of Ser2 antibody was detected in cell layer and sericin layer in region I,and in sericin layer in regions II and III.The Ser3 antibody signal was detected in three regions of the cell layer and the fibroin layer.The structure of M-MSG was similar after HE staining,so only one region of M-MSG was selected for immunohistochemistry.Ser1 antibody and Ser3antibody detected signal in cell layer and sericin layer,while Ser2 antibody did not detect signal.In addition,Ser2 protein was expressed in the front of A-MSG and Ser3protein in the middle and back of A-MSG.4.Sericin gene and sericin analysis of Fl mutant of silkwormThe mutant strain Fl of silkworm has a fluffy multi-layered cocoon shell,which is quite different from the dense cocoon shell formed by normal silkworm.According to previous research reports,the mutation phenotype may be caused by the adhesion layer between the cocoon layers.At present,sericin plays a key role in the formation of cocoon.Therefore,an investigation has been carried out on the mutant Fl of cocoon and the strain 872 of dense cocoon.Firstly,detection was performed at the protein level,and proteomic analysis was performed on the cocoon shells of Fl and 872.The Ser3expression level of Fl was higher than 872 and there was a significant difference.Western blot showed that the signal intensity of Ser3 antibody in Fl was higher than that in 872,which was consistent with the result of proteome.Subsequently,the expression of sericin gene in the silk gland lumen was analyzed at the m RNA level.It was noteworthy that Ser1 was expressed in Fl in A-MSG,but not in 872.Ser3 was expressed in Fl in P-MSG,but not in 872.Finally,we speculated that it might be due to the disorder of Ser1 gene and Ser3 gene expression or the high expression of Ser3 gene that resulted in the loose and multilayer cocoon type phenotype.5.Silkworm sericin antibody to identify different silk and silk fabricsThe later processing of silkworm cocoon mainly includes cocoon drying,silk reeling and degumming.In this process,it is not clear what happens to sericin at each step.In this study,sericin was detected in different cocoon silk production processes.The Qiu?er shell,raw silk,mature silk and white silk of qiuer cocoon were selected for the experiment,and 872?s cocoon were used as the control.The results showed that the signals of Ser1 and Ser3 antibody were detected in 872 cocoon shell,Qiu?er cocoon shell,raw silk and cooked silk.No signal was detected by Ser2 antibody in the above materials.During the process from raw silk to mature silk,the signal of Ser1 antibody decreased a lot.In this process,Ser1 protein was mainly removed.During the process from shell to raw silk,the signal of Ser3 antibody was obviously weakened,which indicated that Ser3 protein was mainly removed in this process,which indicated that Ser3 protein in outer layer was removed first and then Ser1 protein in inner layer was removed in the process of degumming.The signal of Fib-L antibody is gradually increased in shell,raw silk and mature silk.It can be concluded that sericin is continuously removed step by step with the deepening of cocoon processing.In summary,six sericin antibodies were successfully prepared in this study,which can be used not only for the basic research of the secretion process of sericin,but also for the detection of the industrial sericin degumption process.