人TNFα分子N末端、C末端结构的修饰与生物活性的关系

Relationship Between the Structure-modified N-and C-Termini and the Biological Activity of TNFα Molecule

通过对人肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα）──ＴＮＦα分子的Ｎ末端第４、５、１０位以及Ｃ末端第１５７位氨基酸的修饰，由ＴＮＦα原型序列Ｓｅｒ（４）、Ｓｅｒ（５）、……Ａｓｐ（１０）改为Ｃｙｓ、Ｔｈｒ、……Ａｒｇ，Ｃ末端１５７位Ｌｅｕ改为Ｐｈｅ，研究了ＴＮＦα分子结构改变与其生物活性的关系。从ＴＮＦα分子的一级结构和高级结构得知，分子的Ｎ末端可能参与对受体的识别，而Ｃ末端对稳定ＴＮＦα分子的活性形式──三聚体起主要作用，因而将分子修饰部位选在Ｎ、Ｃ末端。采用ＰＣＲ定位突变方法，构建了ＴＮＦα衍生物１０（ＴＮＦαｄｅｒｉｖａｔｉｖｅ１０－ＴＮＦａＤ１０）的表达载体，观察两个部位数个氨基酸改变后对整个ＴＮＦα分子生物活性的影响。实验结果表明：Ｎ、Ｃ末端数个氨基酸的置换并未明显提高ＴＮＦα的表达量，但却提高了其体外细胞毒功能，为原型ＴＮＦα的１０倍左右。原因可能系Ｎ末端第４位由丝氨酸改成半胱氨酸，使ＴＮＦαＤ１０的表达产物呈多聚体状态。ＨＰＬＣ检测示表达产物为三聚体单一峰，在ＳＤＳ－聚丙烯酰胺凝胶电泳还原条件下仍可见到三聚体的蛋白条带为以上推测提供了证据。此外，由于Ｃ末端第１５７位由原型ＴＮ?

In order to elucidate the relationship between N-、C-termini of TNFαand it's biological activity,a TNFαderivative 10(TNFαD10) was prepared by changing amino acid at the N-terminus positions Ser(4)、Ser(5)、Asp(10)and C-terminus position Leu(157) to N-teiminus Cys(4)、Thr(5)、Arg(10)and C-terminus Phe(157)with PCR site-directed mutagenesis.The results showed that the expression level of this mutein has not altered but its cytotoxic activity increased.This might result from trimer formation of TNFαD10 by changing N-terminal Ser(4)residue to Cys.HPLC showed that the molecular weight of TNFαD10 was 17kD,35kD,55kD respectively.In addition,we also found that the stability of TNFαD10 was less than that of TNFα when to be stored at -20℃ for two months.It might be caused by changing Leu(157) to Phe (157).