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目的:探讨溶菌酶N端多肽片段基因的重组并在原核中表达,研究该片段与晚期糖基化终产物(advanced glycation end products,AGEs)的结合作用。方法:从SD大鼠外周血白细胞中抽提总RNA,经逆转录并扩增大鼠溶菌酶N端基因片段,以质粒pET-30 a(+)为载体构建表达质粒pLY77。含pLY77的工程菌Rosetta(DE3)经IPTG诱导,对该重组多肽进行高效表达。表达产物经N i+-NTA琼脂糖柱层析纯化后,得到的溶菌酶N端多肽片段LY77经SDS-PAGE和W estern印迹法鉴定,ELISA法分析体外结合活性。结果:LY77在Rosetta(DE3)中高效表达,主要以可溶性蛋白的形式存在,相对分子质量约为11 000;LY77对体外制备的晚期糖基化蛋白BSA-AGEs具有显著的结合活性。结论:LY77对AGEs具有很高的亲和力,为进一步探讨LY77可能应用于体内促进AGEs的清除和参与免疫调节作用奠定基础。
OBJECTIVE: To investigate the recombination of lysozyme N-terminal polypeptide fragment and its expression in prokaryotic cells, and to study the binding of this fragment to advanced glycation end products (AGEs). METHODS: Total RNA was extracted from peripheral blood leukocytes of SD rats. The N-terminal gene fragment of rat lysozyme was reverse transcribed and amplified. The expression plasmid pLY77 was constructed by using plasmid pET-30 a (+) as a vector. The engineering strain Rosetta (DE3) containing pLY77 was induced by IPTG to express the recombinant polypeptide efficiently. The expressed product was purified by N i + -NTA agarose column chromatography. The lysozyme N-terminal polypeptide fragment LY77 was identified by SDS-PAGE and Western blotting. The in vitro binding activity was analyzed by ELISA. RESULTS: LY77 was highly expressed in Rosetta (DE3) and mainly existed as a soluble protein with a relative molecular mass of about 11 000. LY77 exhibited significant binding activity to BSA-AGEs, an advanced glycation protein prepared in vitro. CONCLUSION: LY77 has a high affinity for AGEs, which lays the foundation for further exploration of the potential role of LY77 in the in vivo promotion of AGEs clearance and participation in immune regulation.