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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?
By TNFα prototype sequence Ser (4), Ser (5), ... ... on TNFα (TNFα) - the N-terminal 4th, 5th and 10th amino acids and the 157th amino acid at the C- Asp (10) was changed to Cys, Thr, ... Arg, and the 157th Leu was changed to Phe. The relationship between the structural change of TNFα and its biological activity was studied. From the primary structure and the higher structure of the TNFα molecule, it is known that the N-terminus of the molecule may be involved in the recognition of the receptor and the C-terminus plays a major role in stabilizing the active form of the TNFα molecule, the trimer, At the N, C terminus. The expression vector of TNFαderivative10-TNFaD10 was constructed by PCR mutagenesis, and the effect of several amino acids changes at two positions on the bioactivity of TNFα was observed. The experimental results show that: N, C terminal amino acid substitution did not significantly improve the expression of TNFα, but increased its cytotoxicity in vitro, the prototype of TNFα about 10 times. The reason may be the fourth N-terminal serine to cysteine, TNFαD10 the expression product was multimer state. HPLC showed that the expressed product was a single peak of trimer, and the protein band of trimer still visible under the condition of SDS-polyacrylamide gel electrophoresis provided the evidence for the above speculation. In addition, since the C-terminal 157th by the prototype TN?