重组人肿瘤坏死因子α衍生物3a突变区的氨基酸序列分析

The amino acid sequencing on mutation region of recombinant human tumor necrosis factor α derivative 3a

目的：对重组人肿瘤坏死因子α衍生物３ａ（ｒｈ－ＴＮＦαＤ３ａ）突变区进行氨基酸序列分析。方法：通过ＰＣＲ扩增，获得５＇端含ｒｈ－ＴＮＦαＤ３ａ突变区的半分子。用融合蛋白表达系统对该半分子进行大肠杆菌表达。表达产物经亲和层析法纯化，用Ⅹａ因子酶切，去除载体多肽，然后进行Ｎ末端２０个氨基酸顺序分析。结果和结论：氨基酸序列符合预想突变序列，即发生下列氨基酸替换：８０位Ｉｌｅ→Ｓｅｒ，９０位Ｌｙｓ→Ｈｉｓ，９２位Ａｓｎ→Ｖａｌ。

Objective: To analyze the amino acid sequencing on mutation region of tumor necrosis factor α derivative 3a(rh TNFαD3a). Methods: PCR was used to produce the TNF half molecule which contains the mutation region in its 5' end. Then, a fusion protein expression system was used to express the TNF half molecule. The fusion protein was purified by affinity chromotography and enzymatically cleaved by Ⅹa factor protease to remove fusion vector peptide. Finally, the N terminal amino acid sequencing was performed. Result and conclusion: The artificial mutation of TNFαD3a was realized as expected: position 80 Ile→Ser, position 90 Lys→His and position 92 Asn→Val.