Purification Of Pupae Lectin And Inhibition Of Tumor Growth By The Extract Of Musca Domestica

Developing functional foods and new drugs to enhance the immunity is one of the main topics in the field of food biotechnology. Musca Domestic is a kind of insect with high immune system. It's easy feeding, rapid reproduce and low cost endow it with bright perspective to be a new source of immune reagent.This paper systematically studied the purification and bioactivity of pupae lectin(MPL) from Musca Domestic. Two Methods of isolation by both glutaraldehydeized red blood cells (GRBC) adsorption and Sepharose-4B affinity column are compared. The active component isolated by the Sephadex G-50 was used to test its anti-tumor activity.The process of purification by GRBC adsorption is following:diluted haemolymph was mixed with 15% GRBC and incubated for 60 min at RT. Then, the mixture was precipitated to get lectin-GRBC. Finally, desorption was carried out using D-galactose solutions. The crude extraction can be purified by Sepharose 4B affinity column. The parameters is as follows: column diameter 1.1 cm, column length 36 cm, adding volume of sample 2ml. Miscellaneous proteins were washed off to A280<0.02 using the BIS buffer and then were washed to A₂₈₀<0.02 using D-galactose solutions. The current velocity was 1 ml/min. Ratio of obtaining by GRBC adsorption is lower than by Sepharose 4B affinity.By SDS-PAGE with 2-mercaptoethanol, the MPL that purified with two methods yielded 3 bands corresponding to 35 Kd, 40 Kd and 70 Kd respectively. It can be identified as lectin qualitatively with coagule ingredients in musca domestica tentatively with Sephadex G- 50 gel column, were usedation test.Peak one and peak two separated from effectiv to the research of antitumor function in vitro. Leukaemia cell K562 was used as experiment tumor cell and cultivated in shell-vial.MTT is adopted to measure the density of half death. The components of two peaks all have certain inhibition on the K562 cell. They all have relations between density and result. Namely with the rising of the density, the sample strengthens the suppressing rate of tumor cell. The inhibition of the component of second peak should be stronger than the first peak. IC₅₀ of the component of first peak is about 100μg/ml, and IC₅₀ of the component of second peak is about 80,Mg/ml.The tumor cell is observed the change of its form under the inversion microscopes. Before and after the effective ingredients acts on K562 cell, the obvious change takes place in its cell form. The cell appears a lot of little round subjects with intact membrane , called apoptosis body.