苏云金芽孢杆菌cry基因在大肠杆菌中表达产物和生物活性

研究了几种Bt cry基因于大肠杆菌(Escherichia coli)中表达产物在pH 10.0的50mmol/L碳酸钠和20mmol/L乙醇胺溶解液中的溶解性 ,发现同样的Cry蛋白在碳酸钠中的溶解度大于乙醇胺。通过胰蛋白酶消化 ,明确Cry1Ca7、Cry1Ia8酶解产物为 38kD多肽 ;Cry1Ie1、Cry1Cb2、Cry2Ab4酶解产物为 41kD多肽 ;Cry1Ac酶解产物为60kD多肽。采用FPLC层析方法对 6种原毒素及其酶解后得到的毒素多肽进行了分离纯化 ,比较了原毒素和毒素的杀虫活性的差异。其结果表明 ,Cry1Ac的原毒素和毒素对棉铃虫初孵幼虫的校正死亡率均为 100% ,Cry2Ab4的原毒素的毒力高于其酶解毒素。     

苏云金芽孢杆菌Cry8Ca2蛋白的纯化与活性的研究

cry8 Ca2基因是本实验室自行分离克隆的一种新型苏云金芽孢杆菌(Bacillus thuringiensis,Bt)杀虫晶体蛋白基因,其表达130 kDa蛋白对铜绿异丽金龟(Anomala corpulenta)和黄褐异丽金龟(A.exoleta)幼虫具有较高的活性。本研究对Cry8Ca2蛋白原毒素及胰凝乳蛋白酶消化片断的纯化条件及活性进行研究,最适的消化条件为质量比1∶20,37℃,消化1 h。通过阴离子交换层析得到纯的活性毒素蛋白。用原毒素与纯化的毒素对铜绿丽金龟幼虫进行生测,Cry8Ca2原毒素蛋白的LC₅₀为0.47μg/g土,纯化的毒素的LC50为0.08μg/g土,毒素的杀虫活性可以达到原毒素的6倍。     

The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.     

由苏云金芽胞杆菌胞晶混合物直接提取原毒素的方法

采用室内实验技术,研究了直接从苏云金芽胞杆菌胞晶混合物提取原毒素的方法。胞晶混合物依次用0.5mol·L-1NaCl和无菌去离子水洗涤3次,洗涤后的沉淀物用0.05mol·L-1NaOH 0.1mmol·L-1PMSF溶液溶解10min,10000r·min-1离心15min去除不溶物。上清液以25%乙酸调至pH4.4,得原毒素蛋白沉淀,冷冻干燥。SDS-PAGE电泳扫描结果表明,原毒素分子量为130kDa,在提取过程中没有发生降解。该提取方法具有操作简便、成本低、效率高等优点。     

Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain

Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene.Because Cry1 Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops.The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments.The media for crystal production and the method for Cry protoxin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin.In this paper, three media for crystal production and the method for Cry1 Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1 Ac protoxin.The results showed that the 1/2 LB(Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1 Ac protoxin in 300 m L 1/2 LB medium was(112.38±5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method.It will be a reference for other Cry protoxin preparation, especially for larger number.     

苏云金芽胞杆菌原毒素在蒙脱石上的吸附特性研究

采用河南信阳产钠型蒙脱石为实验材料,研究了苏云金芽胞杆菌(Bt)工程菌株WG-001原毒素蛋白(130kDa)在粒径为0～0.2μm和0～2μm的蒙脱石上的吸附特性。结果表明,蒙脱石在碳酸盐缓冲体系(pH9)对Bt原毒素蛋白的等温吸附曲线符合Langmuir方程(r2>0.99),当原毒素与蒙脱石的质量比例相同时,蒙脱石的平均粒径越小,单位质量吸附量越高。在磷酸盐体系(pH6～8)pH7时单位质量吸附量最大,在碳酸盐体系(pH9～11)单位质量吸附量随pH的增加而下降。Bt原毒素在蒙脱石上的吸附0.5～1.0h就能达到平衡。随着蒙脱石质量比例的增加,蒙脱石对原毒素的单位质量吸附量减小,但吸附百分率增加。在10℃～50℃范围内,温度对单位质量吸附量影响不大。透射电镜分析表明,吸附原毒素前后蒙脱石的粒径没有明显改变。XRD分析证实,吸附原毒素后蒙脱石层间距没有发生变化。