TAT融合蛋白在SD大鼠肾组织表达的研究

A Study of TAT Fusion Protein Expression in Rat Kindey Tissues

目的 :探讨分子量为 12 0kD的TAT融合蛋白在SD大鼠肾组织的吸收及酶活性的表达情况。方法 :利用“蛋白转导”的方法 ,将人类免疫缺陷病病毒HIV的TAT蛋白与 β -半乳糖苷酶 (β -Galactosidase,β -Gal)相连接 ,构建形成TAT - β -Gal融合蛋白 (分子量为 12 0kD)。将 72只SD大鼠随机分为 3组 (每组n =2 4 ) ,分别腹腔注射TAT - β -Gal、β -Gal和生理盐水 ,于注射后不同时间段取肾组织作病理切片 ,用X -Gal染色分析 ,显微镜下观察肾脏对TAT - β -Gal的吸收及酶活性的表达情况 ,并进行Leica计算机图像分析 ,同时观察肾组织结构改变。结果 :TAT - β -Gal组在经腹腔注射TAT - β -Gal 0 .5h后切片 ,用X -Gal染色 ,显微镜下即可显示着色反应 ,TAT - β -Gal可以快速到达肾组织 ,在肾小球细胞表达酶的活性 ,于 8h达到高峰 ,16h逐渐下降 ;β -Gal组经X -Gal染色后出现极弱的着色反应 ;而生理盐水组 ,经X -Gal染色后无着色反应。结论 :分子量为 12 0kD的TAT -β-Gal融合蛋白可以迅速被SD大鼠肾组织所吸收 ,同时不影响所连接蛋白酶的活性。HIV的TAT蛋白可以作为载体转运大分子 (蛋白或肽类大分子 )物质通过生物膜进入细胞内 。

Objective To probe the condition of absorption and expression of TAT fusion protein (120 kD) in the rat kindey tissues. Methods TAT protein from the human immunodeficiency virus (HIV) was combined with β-Galactosidase (β-Gal) to form the TAT-β-Gal fusion protein (120 kD) by using protein transduction.72 SD rats were randomly divided into three groups (24 in each group).The rats of three groups were respectively given intraperitoneal injection of TAT-β-Gal,β-Gal and 0.9%Natrii chloride. The pathological slices of kindey tissues of each group at different stages were made after injection. We observed the conditions of kindey's absorption TAT-β-Gal and the expression of activity of enzyme under the microscope by using X-Gal stain analysis,then analysed the Leica computer image,and observed the construction of kindey tissues. Results The slices of TAT-β-Gal group 30 min after the intraperitoneal injection were colored by X-Gal. The stain reaction appeared immediately under the microscope. TAT-β-Gal quickly reached the kindey tissues and was absorbed by kindey tissues,and expressed the activity of enzyme in the cells of glomerules which reached the peak at 8h and gradually decreased at 16h;That of β-Gal group weakly appeared to color-reaction after X-Gal-stain. Nothing was appeared in the 0.9% Natrii chloride group . Conclusions TAT-β-Gal fusion protein(120KD) quickly absorbed by SD rats kindey tissues without affecting the protein activity. TAT protein of HIV may be regarded as a transporter trafficking the macromolecule materials across the bio-membrane.It provides experimental basis for the clinical application of macromolecule materials in treatment of diseases.