乳腺注射法表达人组织型纤溶酶原激活剂的研究

Expression of Human Tissue-type Plasminogen Activator(tPA) in Cow via Mammary Gland Injection

目的 :构建tPA乳腺定位表达载体 ,使其在牛乳汁中高效表达 ,观察目的基因表达的规律及其影响因素 ,为建立新型牛乳腺生物反应器提供理论基础。方法 :RT PCR法克隆目的基因 ,通过酶切、连接、分离、纯化等方法构建含tPA cDNA的乳腺定位表达载体 ;采用乳腺注射法将融合基因转入小鼠及牛的乳腺组织中。结果 :乳腺注射外源基因后 ,tPA可在小鼠和牛的乳汁中表达。结论 :乳腺注射法可使目的基因在乳腺组织中稳定地表达较长的时间 ,其表达量与显微注射法没有明显的差异 ,表明外源基因的表达不受转基因方法的影响。但tPA在牛乳汁中的表达量明显高于小鼠的表达量 ,提示不同动物的乳蛋白调控系统有一定的差异 ,可能受着不同的因素或调控系统的影响。

Objective: Expressing tPA in milk by means of constructing a special vector which can only expression in mammary gland,in order to develop a new type mammary gland bioreactor via mammary gland injection. Method: RT-PCR was employed to amplify ht-PA cDNA, and then digestion with the restriction enzymes and was subsequencetly cloned into vector pSP72 for constructing special fusing gene expressed only in the mammary gland. The fusing gene was transfer into the mammary gland tissue of mice and cows. Result:tPA can be expressed in the milk after mammary gland injection. Conclusion:A special vector expressed in mammary gland have been constructed, which have let tPA expressed in milk effectively and there is not significantly difference between mammary gland injection and microinjection. The target gene can expression in mammary gland steadily after mammary gland injection. But the expression level of tPA is dramatically high in cows than in mice, it means that there is some difference of milk proteins regulating system among various animals. Some difference influence may affect and control the expression.