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目的原核表达海蚯蚓蛋白酶基因,探讨其对肿瘤细胞增殖的抑制作用。方法根据环节动物门蚯蚓和单环刺螠丝氨酸蛋白酶c DNA序列多重比对获得的保守序列设计引物,从海蚯蚓肠道组织提取总RNA,经3′RACE和5′RACE技术分别克隆海蚯蚓蛋白酶c DNA序列的3′端和5′端,并进行拼接,对其编码的蛋白质作生物信息学分析;将海蚯蚓蛋白酶成熟肽的编码序列亚克隆至原核表达载体p ET-21a(+),转化大肠埃希菌BL21(DE3)进行诱导表达,采用组氨酸亲和层析柱纯化;MTT法检测重组蛋白的抗肿瘤活性。结果经3′端RACE技术获得约250 bp的海蚯蚓蛋白酶基因c DNA 3′端序列,再经5′RACE技术扩增获得约770 bp的c DNA 5′端序列,拼接可得海蚯蚓蛋白酶c DNA序列全长为880 bp,其编码蛋白含270个氨基酸,具有高度保守的GDSGGP序列;重组蛋白相对分子质量为27 000,主要以包涵体形式表达,约占菌体总蛋白的30%,上清纯化后纯度可达95%;重组蛋白酶对人肿瘤细胞增殖具有明显的抑制作用。结论原核表达了海蚯蚓蛋白酶,可明显抑制人肿瘤细胞的增殖,为抗肿瘤基因工程药物的研发提供了实验依据。
Objective To express prokaryotic protease gene of sea cucumber and explore its inhibitory effect on the proliferation of tumor cells. Methods According to the conserved sequences of the earthworm and monocystorhynchus serpin c DNA sequences, the primers were designed and the total RNA was extracted from the intestinal tissues of the earthworm, and the sea cucumber earth proteases were cloned by 3’RACE and 5 ’RACE techniques c DNA sequence of the 3 ’end and 5’ end, and splicing the encoded protein for bioinformatics analysis; earthworm protease mature peptide subclone the prokaryotic expression vector p ET-21a (+), The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced by histidine affinity chromatography. The antitumor activity of the recombinant protein was tested by MTT assay. Results The 3 ’end of c DNA of sea cucumber earthworm protease gene was obtained by RACE technique at 3’ end. The 5 ’end of c DNA sequence of about 770 bp was amplified by 5’RACE technique. The full length of the DNA sequence was 880 bp, encoding a 270 amino acid protein with a highly conserved GDSGGP sequence. The relative molecular mass of the recombinant protein was 27 000, which was mainly expressed in inclusion bodies, accounting for 30% of total bacterial proteins Purity purity of up to 95%; recombinant protease on human tumor cell proliferation was significantly inhibited. Conclusion Prokaryotic expression of the seaworm protease can significantly inhibit the proliferation of human tumor cells and provide an experimental basis for the development of anti-tumor genetically engineered drugs.