Fiber optic biosensor of immobilized firefly luciferase

Luciferase from firefly lantern extract was immobilized on CNBr-activated Sepharose 4B. The kinetic properties of immobilized luciferase were extensively studied. TheK _m′ for D-luciferin is 11.9 μmol/L, the optimum pH and temperature for Sepharose-bound enzyme were 7.8 and 25°C respectively. A luminescence fiber optic biosensor, making use of immobilized crude luciferase, was developed for assay of ATP. The peak light intensity was linear with respect to ATP concentration in range of 10⁻⁹–10⁻⁵ mol/L. A biological application was also demonstrated with the determination of serum ATP from rats bred in low versus normal oxygen environments.     

萤火虫萤光素酶基因转化花叶芋及表达的研究

将萤火虫萤光素酶基因重组到质粒pBR325中,通过“三亲交配”和同源重组,将萤光素酶基因整合到Ti质粒pGV3850:1103。通过改良的叶盘转化法,用萤光素酶基因转化单子叶植物花叶芋,获得转基因植株。对再生植株的胭脂碱测定、新霉素磷酸转移酶分析、DNA点杂交、萤光素酶基因活性测定,证明萤光素酶基因已转化到花叶芋中并进行了表达。     

SV40早期启动子中原核增强子样序列的初步研究

萤火虫荧光素酶基因cDNA真核表达载体pGL2可在原核细胞中高效表达。该载体中含有SV40早期启动子及荧光素酶基因cDNA5'端总长共758bp的DNA碱基序列，经计算机分析发现，在SV40早期启动子中含有SV40基因组HindⅢB片断中一段长为49bp的DNA序列，且荧光素酶基因cDNA编码区的5'端含有一个原核细胞基因表达调控区相似序列。将pGL2中的荧光素酶基因cDNA（Luc）克隆入真核表达载体pED中，构建成pED-Luc。该载体经表达测定后，只在真核细胞中表达，在原核细胞不中表达。这说明荧光素酶基因cDNA编码区的5'端的原核细胞基因表达调控区相似序列不具表达有活性的荧光 素酶的功能。经DNA序列对比研究，认为pGL2含有的SV40基因组Hind ⅢB片段5123bp-5171bp长49bp的DNA序列可能就是Hind ⅢB片段的原核增强子功能的决定序列。