本文選題：AaIT + CytBa　； 參考：《南方醫(yī)科大學(xué)學(xué)報(bào)》2017年06期

【摘要】：目的 構(gòu)建表達(dá)蝎毒素AaIT或蘇云金桿菌毒素Cyt2Ba的大腸埃希菌,檢測(cè)重組菌對(duì)致倦庫(kù)蚊和白紋伊蚊II齡幼蟲的毒力,并測(cè)試不同劑型的增效作用。方法 分別將AaIT和Cyt2Ba編碼基因克隆于pET28a(+)質(zhì)粒上,并將重組質(zhì)粒分別轉(zhuǎn)化BL21 E.coli獲得分別表達(dá)AaIT和Cyt2Ba的工程菌株,經(jīng)IPTG誘導(dǎo)后,用SDS-PAGE和Western-blot檢測(cè)重組蛋白的表達(dá);測(cè)試不同濃度工程菌菌液對(duì)于蚊幼蟲的殺滅效果;并將有效的工程菌制成干粉劑,檢測(cè)其干粉末及其配劑對(duì)蚊幼蟲的殺滅效果。結(jié)果 AaIT和Cyt2Ba重組蛋白在大腸埃希菌中成功表達(dá),但AaIT重組蛋白對(duì)蚊幼蟲不具有毒性。表達(dá)Cyt2Ba重組蛋白的工程菌菌液對(duì)白紋伊蚊和致倦庫(kù)蚊幼蟲均具有明顯的殺滅作用,48 h的LC50分別為3.00×106和1.25×107cells/m L,其中對(duì)白紋伊蚊幼蟲的殺滅效果要顯著優(yōu)于對(duì)致倦庫(kù)蚊幼蟲的殺滅效果(P0.001),并且表達(dá)Cyt2Ba重組蛋白的工程菌干粉末及其配劑均具有較好的殺蚊幼蟲效果。當(dāng)該工程菌干粉末與干酵母粉、小麥粉和白胡椒粉分別按4∶1、1∶1和1∶4比例制成配劑處理蚊幼蟲48 h后,結(jié)果顯示當(dāng)質(zhì)量比為1:1時(shí),配劑殺蚊幼效果顯著提高(P=0.044),并且白胡椒粉末配劑效果要顯著好于干酵母粉和小麥粉配劑(P=0.002)。結(jié)論 表達(dá)Cyt2Ba的重組大腸埃希菌對(duì)蚊幼蟲具有較好的殺滅作用,合適配劑的使用可以增強(qiáng)其在蚊蟲防制中的效果。
[Abstract]:Objective to construct Escherichia coli expressing scorpion toxin AaIT or Bacillus thuringiensis Cyt2Ba to detect the virulence of recombinant bacteria against Culex pipiens quinquefasciatus and Aedes albopictus II instar larvae and to test the synergism of different dosage forms. Methods the AaIT and Cyt2Ba coding genes were cloned into pET28a () plasmid, and the recombinant plasmid was transformed into BL21 E.coli to obtain the engineering strain expressing AaIT and Cyt2Ba respectively. After IPTG induction, the expression of recombinant protein was detected by SDS-PAGE and Western-blot. The killing effect of different concentrations of engineering bacteria liquid on mosquito larvae was tested, and the effective engineering bacteria were made into dry powder to detect the killing effect of dry powder and its mixture on mosquito larvae. Results AaIT and Cyt2Ba recombinant proteins were successfully expressed in Escherichia coli, but AaIT recombinant proteins were not toxic to mosquito larvae. The LC50 of the engineering liquid expressing Cyt2Ba recombinant protein against both Aedes albopictus and Culex pipiens quinquefasciatus larvae was 3.00 脳 10 ~ 6 and 1.25 脳 107cells/m / L for 48 h, respectively. The killing effect on Aedes albopictus larvae was significantly better than that on Culex pipiens quinquefasciatus larvae. The insecticidal effect of larva was P0.001, and the dry powder and its mixture of recombinant Cyt2Ba protein had better effect on killing mosquito larvae. When the dry powder, dry yeast powder, wheat flour and white pepper powder were made into a mixture of 4: 1: 1: 1 and 1:4 to treat mosquito larvae for 48 h, the results showed that when the mass ratio was 1:1, The effect of the mixture on mosquito control was significantly improved, and the effect of white pepper powder was better than that of dry yeast powder and wheat flour mixture (P0. 002). Conclusion Recombinant Escherichia coli expressing Cyt2Ba has a good killing effect on mosquito larvae, and its effect on mosquito control can be enhanced by the use of suitable compound.
【作者單位】： 南方醫(yī)科大學(xué)公共衛(wèi)生學(xué)院病原生物學(xué)系//廣東省熱帶病研究重點(diǎn)實(shí)驗(yàn)室;
【分類號(hào)】：R184.31

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