螺旋藻的系统分类学及基因工程研究进展

国家863计划资助项目819－04－03号

Preparation and determination of polyclonal antibody to thymosin α1

目的 :制备用于检测转胸腺素基因蓝藻表达产物的特异性抗体。方法 :用提纯的小肽 (2 8个氨基酸 )胸腺素α1与牛血清白蛋白偶联后作为抗原 ,采用皮下多点注射免疫的方法免疫大鼠。经过 3个月的免疫 ,获得抗Tα的多克隆抗体。结果 :用间接酶联免疫吸附 (ELISA)方法 ,测得抗体效价超过 40 96。蛋白印迹 (Westernblot)检测结果显示 ,该抗体能特异性地与胸腺素α1抗原产生明显免疫亲和反应。结论 :所制备的抗体具有很好的灵敏性和特异Objective:To prepare specific antibody against Thymosin α1 Methods:Thymosin α1(Tα1,28 peptide) was conjuncted with BSA as immune antigen,rats were immunized and the polycolonal antibody to Tα1 was obtained.Results:With ELISA detection,the titer of antibody was more than 1:4 096;Western blot analysis showed that the antibody can bind with Tα1 specifically.Conclusion:The prepared antibody against Tα1 has good specificity and reactivity and can be used to detect the expression products of transgenic cyanobacteria which expressed Tα1 gene.国家“863”课题资助!(No 819 0 4 0 3

钝顶螺旋藻化与表达系统的建立

蓝藻是原核生物，遗传背景相对简单，是很理想的真核基因表达受体，其遗传转化研究进展很快。应用穿梭质粒载体和基因整合平台系统已经成功地在多种蓝藻中实现了外源基因的表达。螺旋藻是一类丝状蓝藻，含有大量的蛋白质及其它人体所必需的营养物质,是少数已被确证可以安全使用的蓝藻之一，更适用于作为生产有用蛋白质的生物反应器开发，并制造可直接食用的基因工程产品。但是，螺旋藻作为外源基因工程受体的研究由于缺乏行之有效的遗传转化途径而始终进展缓慢。 螺旋藻内高活性的胞内核酸酶是外源基因转化螺旋藻的主要障碍。我们以供体质粒pEUTISI为主要的酶消化底物，通过多种方法处理螺旋藻细胞，然后检测其胞内核酸酶初提液对pEU...Spirulina platensis, a kind of cyanobacterium, is famous for its rich content of protein and other sources of nutrients. In these years, it has been rapid progresses on genetic engineering of other cyanobacteria, but Spirulina platensis is extremely hostile to foreign gene transformation. The active endocellular nuclease of Spirulina platensis is believed to be the most serious barrier of foreig...学位：理学硕士院系专业：生命科学学院生物学系_植物学学号：19982600

REPRESSION OF ENDOCELLULAR NUCLEASE ACTIVITY IN Spirulina platensis

螺旋藻细胞内较高的胞内核酸酶活性是外源基因转化螺旋藻的主要障碍。以供体质粒pEUTISI为酶消化底物 ,通过多种方法处理螺旋藻细胞 ,然后检测其胞内核酸酶粗提液对pEUTISI的降解作用 ,结果表明 ,2mmol/L以上的EDTA处理16h或无Mg2 +螺旋藻培养基培养72h以上 ,都可使处于对数生长期的螺旋藻胞内核酸酶活性显著降低 ;低于28℃(如24℃)培养也可降低螺旋藻的胞内核酸酶活性。根据实验结果 ,建议在螺旋藻转化前72h开始低温、无Mg2 +培养 ,转化前16h提高培养基中EDTA的浓度至2mmol/L,就能获得胞内核酸酶活性极低的受体藻 ,有利于外源基因的转化。The high-activity endocellular nuclease of Spirulina platensis is believed to be the most serious barrier to foreign gene transformation.We detected the status of plasmid pEUTISI after incubation with endocellular nuclease extract from Spir-ulina platensis.Results showed that the activity of endocellular nuclease could be efficiently repressed when the Spirulina platensis was cultured in the medium of EDTA at the concentration of2mmol/L above16hours,or cultured in Mg 2+ -free mediumfor more than72hours.When the Spirulina platensis was cultured in lower temperature,such as24℃,the enzyme activity also showed a little decrease.By these results,it suggests that Spirulina platensis be cultured in Mg 2+ -free medium at24℃for72hours,and improve the EDTA concentration to2mmol/L for16hours ahead of transformation.Then the strain of Spirulina platensis contained lowactivity of endocellular nuclease,which were suitable for transformation,could be obtained.国家863计划资助项目819 04 03号;; 福建省自然科学基金资助项目B0210001

Expression of Thymosin α_1 Gene in Spirulina platensis

将钝顶螺旋藻 (Spirulinaplatensis)在 2 4℃培养 ,并经 2mmol/LEDTA预处理16～ 2 4h ,以本实验室构建的基因整合平台系统供体质粒 pEUTISI进行超声波转化螺旋藻 ,经筛选获得了具G4 18抗性的转化藻株。通过PCR扩增和Southern杂交证实 ,pEUTISI中目的基因UB Tα1和nptII基因已整合到钝顶螺旋藻染色体上。转化藻株经4 5℃热诱导 4 0min后 ,进行蛋白质SDS PAGE电泳和Westernblot,杂交结果证实 ,外源胸腺素α1基因在螺旋藻中得到有效表达。By ultrasonic transformation, donor plasmid pEUTISI of gene intergration platform system was successfully introduced into Spirulina platensis which had been cultured at 24℃ and treated with 2mmol/L EDTA for 16～24 hours. Screened by G418, the transformants which were integrated the foreign DNA were selected. PCR and southern blotting data show that the foreign DNA including nptII gene and UB-Tα 1 gene has been integrated into Spirulina platensis chromosomal DNA. Further confirmation was tested by SDS-PAGE and western blotting after the transformants were heat shock induced at 45℃ for 40 min. The results proved that the foreign UB-Tα 1 gene has been effectively expressed in transgene Spirulina platensis.86 3计划 ( 86 3- 819- 0 4- 0 3);; 福建省自然科学基金 (B0 2 10 0 0 1)资助项目