Purification,Identification,and Functional Analysis Of The Novel Immunomodulatory Peptides From Silkworm Pupa Protein

Silkworm chrysalis has been a kind of food that nourishes and strengthens the body,milds the spleen and kidney since ancient times.It has high nutritional and medicinal value.Silkworm pupae is rich in protein and essential amino acids,vitamins and trace elements for human body.It is a kind of high-quality protein raw material.Our country can produce more than 700,000 tons of fresh silkworm chrysalis every year.Most of them are treated as animal feed,which has not been rationally utilized.Studies have shown that proteins can be degraded by enzymes to produce a large number of small molecule peptide segments,and most of the proteins consumed by human beings are digested and absorbed in the form of low-peptide after digestion by digestive enzymes.Some of these polypeptide fragments can significantly improve the body's life activities,such as promoting digestion and absorption,enhancing immunity,regulating hormone secretion and so on.These polypeptides are called bioactive peptides.In order to expand the utilization of silkworm chrysalis,biological active peptide was prepared from silkworm chrysalis as protein raw material.The protease solution of silkworm pupa was obtained by enzymolysis of alkaline protease and assisted by ultrafine grinding or ultrasonic wave.The immune peptides of silkworm pupa protein were separated by gel chromatography,and the amino acid sequence of its immunopeptide was identified by mass spectrum.The biological activity was verified by oxidation test,the stability of the immunopeptides were evaluated by simulated gastrointestinal test in vitro,and the mechanism of action of the immunopeptides were explored by detecting the expression of the related immune factors in mouse spleen cells.The following results were obtained:Silkworm pupa protein was obtained by alkali extraction after degreasing with petroleum ether.Silkworm pupa proteins were subjected to ultrafine crushing with alkaline protease enzymatic hydrolysis(ultramicro-enzymatic hydrolysis)and ultrasonic crushing with alkaline protease enzymatic hydrolysis(ultrasound-enzymatic hydrolysis).The proliferation rate of spleen cells in mice were 87.5%,88.35% and 88.9% at the help of the three kinds of enzymatic hydrolysates in concentration of 100 ug/mL respectively.Dextran gel Sephadex-G100 was used to separate micronization-enzyme hydrolysate and ultrasonic-enzyme hydrolysate respectively.The components that showed the better activity to promote the proliferation of splenocytes were selected and further separated by Sephadex-G15.The highest rate of splenocytes proliferation in mice was 90.3% and 91.1% respectively.The two components were identified by high performance liquid chromatography-mass spectrometry and the results as follows: ultramicro-enzymatic hydrolysis: DHAV,GN and PNPNTN,with PNTNTN was the main component;ultrasound-enzymatic hydrolysis: DHAV,NGMGK/GNMGK,NGYSK/GNYSK and NPRVNG/NPVRNG,with DHAV was the main component.The test of stability and bioactivity for DHAV peptide showed that the immune activity of DHAV peptide did not change significantly after treated with pepsine(1%,W/W)and trypsin(1%,W/W)for 4 hours.The spleen lymphocyte induction experiment showed that DHAV could effectively promote the proliferation of B lymphocyte and T lymphocyte in mice.In the preliminary study on the mechanism of immune regulation,the expression of immune-related protein factors(IL-6,IL-12,NF-kappa B,Cyclin D1,CDK4)were detected by fluorescence quantitative polymerase chain reaction(q-PRC)in murine spleen cells interfered with silkworm pupa protein immunopeptide.The results showed that Cycling D1 and CDK4 were up-regulated 2.08 times and 2.11 times respectively,and NF-kappa B was up-regulated 6.78 times in the experimental group.Immunopeptide from silkworm pupa protein could induce the activation of NF-kappa B,transcription could regulate the expression of Cycling D1 and CDK4 to promote the transformation of cells from G0/G1 phase to S phase,thus the cell cycle was shorten and the proliferation rate of spleen cells was increased.On the other hand,IL-12 in the experimental group was 13.27 times higher than that in the control group.IL-6 increased only 7.29 times.Immunopeptide from silkworm pupa protein may play an immunoregulatory role by promoting the balance transformation of Th cells to Th1 type.