灵芝lz8基因的克隆和原核表达及LZ-8蛋白的免疫印迹检测

Cloning and prokaryotic expression of lz8 gene and immunoblotting detection of LZ-8,an immunomodulatory protein from Ganoderma lucidum

根据已报道的lz8基因序列设计引物,以灵芝基因组DNA为模板,PCR扩增获得lz8基因。构建了原核表达载体pET30a-lz8,转化原核表达宿主菌RosettaDE3,IPTG诱导融合蛋白表达,并用Ni-NTA亲和层析柱对LZ-8蛋白进行分离纯化。将纯化的LZ-8蛋白用Freund佐剂乳化后注射到新西兰白兔体内,经数次加强免疫后采血分离抗血清,并以抗血清为探针建立了LZ-8蛋白的免疫印迹法定性检测方法。

The detection of LZ-8, an immunomodulatory protein from Ganoderma lucidum, with immunoblot was investigated. The DNA encoding LZ-8 was cloning by PCR using the total DNA of Ganoderma lucidium as template. The lz8 gene was inserted into plasmid pET-30a and transformed into Rosetta DE3. LZ-8 was purified by Ni-NTA from the lysate of bacteria with pET3Oa-lz8 after IPTG induction. Purified LZ-8 emulsified with Freund＇s adjuvant was injected subcutaneously into New Zealand white rabbits. After several immune enhancements, these animals were bled and sera were separated. Antiserum against LZ-8 in western blot was applied to determine the presence of LZ-8.