Molecular Cloning And Expression Analysis Of The Peptidoglycan Recognition Protein-B From The Cotton Bollworm Helicoverpa Armigera

Peptidoglycan recognition protein is an important pattern recognition receptor and play an important role in the insect innate immune response. Combined with peptidoglycan can activate the prophenoloxidase cascade, induct phagocytosis and mediate signal transduction in Toll and the Imd pathway.On the specific features of PGRP in insect innate immune system in the Drosophila research more, but research is also relatively rare in the Lepidoptera, about its function in the innate immune response is not very clear. Based on the cotton bollworm, Helicoverpa armigera as the research object, cloning a PGRP gene, named PGRP-B in a preliminary study of innate immune function of cotton bollworm. The results are as follows:1. HaPGRP -B gene cloning and sequence analysis:Cloning PGRP gene obtained the length of 707bp cDNA, its ORF is a complete open reading frame, coding 198 amino acids, predict protein for 23Da, theoretical molecular weight for a isoelectric point5.67. The protein structure domain analysis shows that there are two structure domains, one is a PGRP domain, located in 35 amino acids to a 175 amino acids from the N end; the other is a Ami2 domain, which is the N-terminal 43 amino acids to 181 amino acids.Sequence alignment shows the PGRP gene sequences of amino acids and Samia Cynthia ricini similarity of PGRP-B up to 84, and Manduca Sexta PGRP-2 and Ostrinia nubilalis similarity of PGRP-B up to 83 percent and 78 percent respectively, So we will clone the gene is named HaPGRP-B. Constructing evolutionary trees indicate:homology of HaPGRP-B and Lepidoptera PGRP-B highest consistency is classified as a family, but unlike Lepidoptera genetic relationships of the original PGRPs far, their opposition is classified as a family.2. Tissue specific expression analysis:use semi-quantitative RT-PCR method, HaPGRP-B gene to transcribe the different organizations in cotton bollworm was studied. The results showed that in fat body, midgut, hemolymph in three kinds of tissues, and expression in fat in the fat body of the highest amount, epidermis express don’t express.3. HaPGRP-B expression changes after injection of different types of microorganisms using fluorescence quantitative PCR methods for detection of the cotton bollworm after injection of different microbial gene expression changes of PGRP-B in the fat body. Results:injection of Staphylococcus auras 6h and 12h,HaPGRP-B expressions after significantly raised; injection Saccharomyces cerevisiae expression 6h,HaPGRP-B after significantly raised; and injection of Escherichia coli HaPGRP-B is not significant changed in expression. These results showe that HaPGRP-B as a pattern recognition receptor, likely to identify Gram positive bacteria and fungi, and does not identify gram-negative bacteria.4. The prokaryotic expression: design-specific primers, using RT-PCR amplification are HaPGRP-B full length, and then clone it to prokaryotic expression vector pET-30a (+) to the construction of recombinant expression plasmid HaPGRP-B-pET-30a (+) in Escherichia coli BL21 (DE3) strains in order to successfully try expression, laid the foundation for subsequent research.