Studies On Polymorphism And Gene Cloning Of Chymotrypsin Inhibitors From Haemolymph Of Eri Silkworm, Philosamia Cynthia Ricini

Protein inhibitors of proteinases are ubiquitous. They present in multiple forms in numerous tissues of animals and plants, as well as in microorganisms. Their primary physiological function is the prevention of unnecessary proteolysis. Chymotrypsin inhibitor (CI) is one class of serine proteinase inhibitors, which play a major role in regulating the proteinase activity in the insect haemolymph. CI is essential to life, and multiple forms of CI are major factors in insect innate immune system. Eri silkworm, Philosamia cynthia ricini, is one of the three major spun silk insects in our country. The investigation on chymotrypsin inhibitors in their haemolymph will benefit to elucidate the physiological and biochemical process and relative research on other lepidopteran insects.Using the method of acid and alkaline native polyacrylamide gel-electrophoresis followed by activity staining, the distribution of CI polymorphism in twenty strains of eri silkworm and the changes of CI activity in different developmented stages were investigated. Based on the different isoelectric points resulted in different migration rates, 7 types of Cls were detected by alkaline native-PAGE, which were designated as E-CI1, E-CI2, E-CI3, E-CI4, E-CI5, E-CI6 and E-CI7, respectively. Two types of CIs were detected by acid native-PAGE, which were designated as E-CIa and E-CIb. Among the detected CIs, 7 types including E-CI1, E-CI2, E-CI3, E-CI6, E-CI7, E-CIa and E-Clb existed in all tested strains, suggesting that they are essential to normal physiological actions of eri silkworm larvae. In different developmental stages the actimeity of CIs from the same strain revealed that Cls gradually increased along with the development of larvae in the fifth instar.The specific primers were desigened based on the sequence of cDNA encoding a mature Cl-bl in silkworm, Bombyx mori. The whole RNAs from 8 strains of eri silkworm were used as templates. E-CI genes were cloned by RT-PCR method. The sequence analyses of the 8 cloned E-C1 genes showed that they were the same gene and 189bp in length, which encodes a putative mature protein of 62 amino acid residues. Prediction of protein profile reveaks this protein harbors a Kunitz domain. Cluster W alignment indicated that the nucleotide sequence of the cloned gene include the sequence of Bombyx mori which encode mature CI-bl.Then the E-CI was subcloned to pET28a, the prokaryotic expression plasmids. The recombinant expression plasmid was constructed to express the recombinant protein in E. coli BL21. The result of SDS-PAGE indicated that the expressed protein is presented in the form of inclusion bodies. MW of the fusion protein induced was about 15 kD and the optimum inducing period was 8 hours.