The Immunological Function Of Hemolin And Its Interacting Protein Yippee In Antheraea Pernyi

Antheraea pernyi is economically important wild silk producing insect.It is commercially raised for the production of silk that is used as a raw material in textile industry.Furthermore,it is greatly important in pharmaceutical industry as well as it is a rich source of nutritious food,containing all essential amino acid required for human body.Hence,due to its high economic importance,there is dire need to conduct immunological studies,particularly the role of genes in the innate immune defense of A.pernyi.In the present study,we cloned Hemolin(Ap-Hemolin)and Yippee(Ap-Yippee)genes from A.pernyi,and evaluated their functions in the immune responses,further we investigated the interaction mechanism involved between them.The results of this study include the following five aspects: 1.Cloning and expression of Ap-Hemolin and Ap-Yippee genes.We designed primers to amplify Ap-Hemolin and Ap-Yippee genesfrom known sequence of silkworm and other lepidopteran species.Polymerase chain reaction was performed to amplify Ap-Hemolin and Ap-Yippee genes,and the obtained products were sequenced.The fragment of Ap-Hemolin gene was 1418 bp in size,including 41 bp 5'-untranslated region and 135 bp 3'-untranslated region.The open reading frame(ORF)contained 1242 bp encoding 413 amino acids.Phylogenetic analysis showed that Ap-Hemolin belongs to the Hemolin family.The Ap-Yippee gene consists of 366 bp open reading frame,encoding 121 amino acids.It belongs to the Yippee-Mis 18 superfamily with a potential zinc finger domain.The ORF of Ap-Hemolin and Ap-Yippee was inserted into the Pet-28 a + expression vector and transformed into E.coli.The recombinant proteins were expressed and then purified using a nickel affinity column.rabbit polyclonal antibodies were preparedto further confirm the function of Ap-Hemolin and Ap-Yippee.2.Spatiotemporal expression distribution of Ap-Hemolin and Ap-Yippee.We examined the expression of Ap-Hemolin and Ap-Yippee in different tissues of three-day-old tussah larvae.The results showed that Ap-Hemolin and Ap-Yippee were ubiquitously expressed in all tissues.The Ap-Hemolin expression level in haemolymph and fat body was higher,while Ap-Yippee expression level was higher in fat body and malpigian tubules.Further we determined the developmental profile of Ap-Hemolin,the transcript expression was significantly higher in pupal and moth than other other stages,whereas Ap-Yippee was higher in ovary and pupal stages.3.Relationship between Ap-Yippee and Ap-HemolinWe used Ap-Hemolin and Ap-Yippee polyclonal antibodies to detect the interaction between Ap-Hemolin and Ap-Yippee proteins in vitro using Far-Western methods.The results revealed that Ap-Hemolin and Ap-Yippee proteins could bind to each other in vitro.Further,the silencing of AP-Hemolin decreased the expression of AP-Yippee,whereas the knock-down of AP-Yippee also showed similar results.Indicating that there is a certain relationship between the AP-Hemolin and AP-Yippee,and might other genes are involved the regulation process.4.Immunological Functional Analysis of Ap-Hemolin and Ap-Yippee Proteins.(AP-NPV),Escherichia coli(E.coli)and Beauveria bassiana(B.bassiana)were used to study the expression of Ap-Hemolin and Ap-Yippee in hemolymph and fat body.Both the Ap-Hemolin and Ap-Yippee transcript and protein levels were up-regulated following microbial stress.The results showed that Ap-Hemolin and Ap-Yippee play an important role in the immune response of A.pernyi.In vitro agglutination test found that the recombinant Ap-Hemolin protein in vitro is still active and can play a role in the agglutination of E.coli.