Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Sericulture > Silkworm basic science > Silkworm physiology, genetics,ecology, biology,physics, biochemistry > Silkworm biochemistry

Study on the Function of Hemolymph Protein Bmhp20 in Silkworm, Bombyx Mori

Author LiuBiLang
Tutor XiangZhongHuai;HeNingJia
School Southwestern University
Course Genetics
Keywords Silkworm Immune response Microbial combination Black reaction Bmhp20
CLC S881.24
Type PhD thesis
Year 2011
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The insects living environment is very special, and the reproduction of the population in order to survive, insects formed in the course of long-term struggle with pathogens strong immune system. Insect innate immune system, it is characterized by the body pattern recognition receptors to identify exogenous pathogens conservative, and thus cause humoral and cellular immunity. The synthesis of antimicrobial peptides with antibacterial effect belongs humoral immunity and cell-mediated immunity, including phagocytosis and parcel role. Pathogen recognition protein has an important role in the innate immune system of the insect. Peptidoglycan recognition proteins recognize peptidoglycan can lead to the generation of antimicrobial peptides glucan recognition protein identification glucan can also cause similar physiological responses. Apart from these two studies is more clearly identify the protein family, there are other types of recognition proteins in the immune response of the insects on the outside also plays an important role. CIb1 is purified from the silkworm body fluids having chymotrypsin inhibitory activity of the Kunitz-type protease inhibitors. Recent studies indicate that CIb1 involved in the immune response of the silkworm, a lipopolysaccharide binding activity. Speculated that it may signal transduction mediated stimulation of xenobiotics. The purpose of this study is to learn more about CIb1 in the the silkworm body of the function, the first through the interaction of the protein for its binding proteins in body fluids. According to the N-terminal amino acid sequence, was cloned encoding the protein of the full-length cDNA (named Bmhp20). By in vitro expression purification Bmhp20 protein, and its function. The key findings are as follows: 1. Silkworm serine protease inhibitor CIb1 purified and humoral protein interactions CIb1 of small molecule is alkaline chymotrypsin inhibitor, in vitro inhibition of the activity of chymotrypsin. Purification strategy developed according to these characteristics to be monitored in the purification process using activity of the chymotrypsin inhibitor. First by ammonium sulfate precipitation, and removing the part of the hybrid protein. According to the molecular weight and isoelectric point of the CIb1 was purified by gel filtration chromatography and ion exchange. The activity staining and purified by mass spectrometry to identify the CIb1 having the activity of a single protein. CIb1 labeled with biotin binding reaction with the body fluids of the silkworm. Far-Western the results display CIb1 untreated silkworm humoral protein interaction; silkworm CIb1 lipopolysaccharide-stimulated humoral protein interacts. 2.CIb1 prokaryotic protein expression, purification and humoral protein interaction coding CIb1 the mature protein cloned into pDEST17 in prokaryotic expression. The plasmids were transformed into the Rosetta the four prokaryotic expression strain Origami B (DE3), BL21 (DE3), JM109 (DE3) after induction expression. CIb1 having three disulfide bonds chymotrypsin inhibitor, the expression of the protein to be able to in the prokaryotic system, the system correctly folded in order to conduct further experiments. Chymotrypsin inhibitor activity staining screening trxB and gor double mutant origami B (DE3) to express active recombinant the CIb1 the protein (rCIb1). rCIb1 exists mainly in the form of inclusion bodies in origami B (DE3). Will be expressed in the form of inclusion bodies rCIb1 urea solubilization S 200 gel column purification and refolding. Activity staining that purified rCIb1 protein active. The preceding experiments have proved that the CIb1 with induced the silkworm body fluids after the protein interaction. In the pull down assay in the rCIb1 and LPS-induced 24 hours after the silkworm body fluids, rCIb1 occurred in the Ni-NTA column with body fluids protein the mutual binding reaction. With rinsing buffer wash with Ni-NTA non-specific binding proteins, with a high concentration of imidazole in the elution buffer after elution was carried out and found that the elution of high molecular weight proteins can be used CIb1 antibody hybridization chromogenic. Speculated here may be CIb1 and silkworm humoral protein complex formed, this composite body having SDS-stable. Eluted by electrically separate proteins and do N-terminal sequencing. The N-terminal 20 amino acids of the protein sequencing analysis showed that the protein HGSSDVDGSGEVEAVAGTLK, no previous studies. The full-length 3.CIb1 combined protein Bmhp20 coding gene cloning and expression analysis of protein N-terminal sequence HGSSDVDGSGEVEAVAGTLK the silkworm database tBLASTn search a predict gene BGIBMGA002175 sequence match. EST sequence, primers were designed based on the gene was cloned and sequenced to verify the gene, the verification result shows the sequencing of the gene sequence with the binding protein sequence is consistent to prove that the cloned gene is the gene sequence of the binding protein. Gene 5 'end of clone to obtain the full-length sequence. The gene is 740 bp, 645 bp, an open reading frame encoding a 215 amino acid peptide. The region of the N-terminal 18 amino acid signal peptide, the predicted molecular weight of 21.5 kDa and an isoelectric point of 5.5. Exists in the form of the molecular weight of the binding protein and protein the named Bmhp20 (GenBank number: GU015849). Bmhp20 gene consists of three exons and two introns constitute. The gene sequence encoding the mature protein is an outer at the last exon on. NCBI BLAST analysis showed with Bmhp20 similar to the amino acid sequence does not exist in other species. The analysis found that the amino acid sequence composition, Bmhp20 mainly by a few amino acids constitute 26.9% of the total amino acids glycine, serine accounted for 13.2%, 12.2% phenylalanine, aspartic acid 10.7%, 9.1% histidine . In ELM database analysis found, Bmhp20 rich glycosaminoglycan binding sites (DGSGE DNSGF NSGF HSGF DSGF DSAL). Bombyx mori fifth instar three-day tissue expression microarray analysis showed no sexual difference Bmhp20 gene expression in the head, body wall, fat body and ovarian four organizations expressed. This result is consistent with the RT-PCR analysis Bmhp20 expression RT-PCR analysis of the Bmhp20 gene expression throughout the developmental stages in the silkworm Bmhp20 genes highly expressed throughout the larval stage of the silkworm has. The high expression level of the first day on Cocooning after suppressed. In pupation fourth, five days appeared to peak a high scale. Since CIb1 participation silkworm's immune reaction, while CIb1 Bmhp20 the outer the xenobiotics stimulation interact speculated Bmhp20 might also be involved in immune response. The silkworm E. coli, the black-breasted septicemia Bacillus bassiana stimulus, quantitative RT-PCR analysis of mRNA expression in the fat body of Bmhp20. Compared with the control, and 1 hour after injection of E. coli, the mRNA of the Bmhp20 rapid rise, and then expression was inhibited. Stimulation of the bassiana expression of Bmhp20 is rising rapidly in 3,6 hours suppressed expression took place in 12 hours when raised. Impact of the largest black-breasted Bmhp20 expression septicemia bacteria, impact it Bmhp20 the to be higher than the other two bacteria, and within 24 hours maximum. The the Bmhp20 gene Bacillus black chest septicemia strong reaction may imply that the genes in the silkworm major bacterial septicemia pathogen (the black chest septicemia Bacillus) should play an important role. 4. His-Bmhp20 prokaryotic fusion protein expression, purification and antibody preparation encoding mature Bmhp20 protein cloned into pET28a vector, Bmhp20 gene has a large number of rare codons, selection Rosetta strain expressed. Inducible protein composition analysis showed that the Bmhp20 protein in Rosetta strain can be expressed in a soluble form, which is conducive to the follow-up study of protein function. The expressed protein with a hexahistidine fusion tag using Ni-NTA resin affinity purification, and then the sample for further purification by Superdex 75 gel column. Bmhp20 amino acid composition of the protein has a large amount of histidine, the labels need to eliminate the influence of hexahistidine fusion tag may cause experimental excision. With a label protein His-Bmhp20 in purified by thrombin digestion, digestion after thrombin residues removed with streptavidin Agarose, a hexa-histidine tag cut using Superdex 75 gel filtration chromatography to remove, to give the pure Bmhp20 . Bmhp20 immune adult male rabbits, polyclonal antibody, the detected titer reached 1:100000 serum antiserum and purified, and stored at -30 ℃ until. The 5. Bmhp20 function analysis the 1) Bmhp20 secretory protein synthesis in the amino acid sequence analysis of the silkworm fluids Bmhp20 having glycosaminoglycan binding sites may be involved in the immune recognition of the silkworm. The Bombyx mori fifth instar larvae for the three days of the organizations the protein Bmhp20 immunoblotting Bmhp20 signal only in the silkworm body fluids, and other organizations have been inspected less than Bmhp20. The the same time Bmhp20 is identified in the silkworm body fluids interacting proteins, these indicate that the Bmhp20 silkworm body fluids and protein function with CIb1. The 2) Bmhp20 does not have casein hydrolyzate function Bmhp20 with the chymotrypsin inhibitor CIb1 can be combined with each other, first consider Bmhp20 in the silkworm might as hydrolase and regulation of the physiological response of the silkworm, its reaction control by CIb1 degree. In vitro testing of the activity, Bmhp20 results show that Bmhp20 does not have the activity of the hydrolyzed casein. Speculated Bmhp20 is not an enzyme, but in other forms involved in silkworm on the outside of the immune response. 3) Bmhp20 protein content of body fluids in E. coli after stimulation reduces mori black chest septicemia Bacillus coli, after stimulation bassiana, collected by centrifugation body fluids, to detect the presence of Bmhp20 in body fluids. Western blotting results showed the the silkworm by the black chest septicemia Bacillus bassiana after stimulation, the body fluids Bmhp20 content little change. E. coli after stimulation, however, Bmhp20 content decreased with increasing stimulation time. Analyze the reasons may be because detection is centrifugal the silkworm body fluids after protein due the CIb1 combined with E. coli, speculated in lipopolysaccharide-stimulated the after CIb1 and Bmhp20 produce interaction cause the humoral free Bmhp20 no timely supplement reduction in the amount of protein. 4) Bmhp20 polysaccharide capable of binding in vitro and Gram positive bacteria, and fungi in vitro binding experiments show Bmhp20 in vitro with chitin, cellulose, curdlan combination. Bmhp20 and Gram-positive bacteria (Staphylococcus aureus, black chest septicemia Bacillus)-negative bacteria (Serratia marcescens, Pseudomonas aeruginosa) and fungi (Beauveria bassiana, Pichia) binding experiments show that Bmhp20 combined with Gram-positive bacteria and fungi. This result implied Bmhp20 silkworm immune response may play a pattern recognition of the role. 5) Bmhp20 not participate in the phagocytosis Bmhp20 protein with FITC-labeled S. aureus and B. bassiana after incubation in vitro observation Bmhp20 whether silkworm blood cells will promote phagocytosis of Staphylococcus aureus and B. bassiana. The results show that Bmhp20 do not have to promote cell phagocytosis. Will take blood cells after 1 hour of FITC-labeled S. aureus and Beauveria injection silkworm observed under a fluorescence microscope, the blood cells of the FITC-labeled bacteria produce phagocytosis unrelated but with Bmhp20. The in vitro and in vivo experiments proved single Bmhp20 protein does not participate in phagocytosis. The 6) Bmhp20 participate in the inhibition of the silkworm blackening reaction the silkworm larvae humoral reagent (SLP) is a detection reagent with the silkworm body fluids made. Add glucan or peptidoglycan reaction can cause blackening. SLP reagent blackening inhibition experiments showed that compared to the black reaction caused by direct stimulation of glucan or peptidoglycan the previously added Bmhp20 could significantly inhibit the occurrence of blackening of the reaction. The this results prove Bmhp20 having inhibiting the silkworm blackening reaction.

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