人α-防御素-1(α-HNP-1)基因乳腺定位表达载体的构建及表达

目的:将人α-防御素-1(α-HNP-1)基因与山羊β-乳球蛋白(Beta-Lac-toglobulin,BLG)基因5’调控区序列融合,构建α-HNP-1基因的乳腺定位表达载体并对其进行检测。方法:用PCR方法获得α-HNP-1基因片断并克隆于pMD18-T载体,经DNA测序证实α-HNP-1基因序列无误后,再将其亚克隆到真核表达载体pcDNA3.1(+),构建pcDNA3.1-HNP-1重组质粒。用PCR方法扩增出BLG基因5’区调控序列并克隆到pcD-NA3.1-HNP-1中,构建乳腺定位表达载体pcDNA3.1-BLG-HNP-1,该载体经脂质体包裹后,经乳腺导管注入妊娠中后期大鼠乳腺中进行表达。结果:经酶切鉴定和DNA测序分析证实重组质粒载体pcDNA3.1-BLG-HNP-1构建正确,经ELISA检测,大鼠乳汁中有α-HNP-1表达,其48h表达量为27.67ng/ml,72h表达量为49.00ng/ml。结论:成功构建了α-HNP-1基因乳腺定位表达载体并最终在乳腺获得一定量表达,说明乳腺导管注入法是一种较理想的评价乳腺特异表达载体的可行性方法。

Construction of Mammary-Specific Expression Vector of Human α-Defensin-1 (α-HNP-1) Gene

Objective: To construct a mammary-specific expression plasmid which containing beta-lactoglobulin (BLG) 5' regulatory sequence and human α-defensin -1 (α-HNP-1) gene,and detect its feasibility.Method: By PCR and T-A cloning techniques,α-HNP-1 gene from human peripheral blood was amplified and cloned into pMD18-T vector, then the recombinant plasmid was digested with EcoR I and Xho I,and subcloned into plasmid pcDNA3.1(+) to construct recombinant plasmid pcDNA3.1-HNP-1.After that, BLG gene promoter amplified from goat peripheral blood was digested with Kpn I and EcoR I and cloned into pcDNA3.1-HNP-1 to construct mammary-specific expression vector pcDNA3.1-BLG-HNP-1. The vector mixed with liposome was injected into mammary gland of mid-term and later stage of pregnant rats through mammary gland center duct.Results: Restriction enzyme digesting and DNA sequencing confirmed that the recombinant expression plasmid (pcDNA3.1-BLG-HNP-1) had been constructed correctly.ELISA confirmed the expression of α-HNP-1 in rats milk.Conclusion: A mammary-specific expression vector pcDNA3.1-BLG-HNP-1 is constructed successfully and it can express in rats milk.This shows that mammary gland duct injection is an ideal way that can assessed the feasibility of mammary-specific expression vector.