研究了培养基、温度、诱导时间、IPTG和氨苄青霉素终浓度以及诱导前后温度变化等不同条件对重组菌菌体生长和融合蛋白表达量的影响,以期获得猪囊尾蚴排泄分泌抗原(Es Ag)Ts8 B1蛋白基因在大肠杆菌中的最大表达量.结果显示,使用TB培养基于37℃培养3 h后,采用终浓度为0.2mmol/L的IFrG和200mm0L/L的氨苄青霉素在32℃过夜诱导培养,pGEX-6p-1/TsSBl融合蛋白表达量最大,表达的融合蛋白约占菌体总蛋白的33%.SDS-PAGE表明p(;EX-6p-1/Ts8Bl融合蛋白大小约为33 kDa,与预计的分子量大小一致,Western blot分析表明其能与猪囊尾蚴多克隆抗体起特异性反应,并获得最大产量的融合蛋白.为研究其在猪囊尾蚴病检测中的应用价值奠定了基础.
王秋霞, 张美英, 张改平, 王选年, 宁长申, 鲁琨, 张龙现, 菅复春
. 猪囊尾蚴排泄分泌抗原Ts881蛋白原核表达条件的优化[J]. 华北农学报, 2009
, 24(3)
: 59
-63
.
DOI: 10.7668/hbnxb.2009.03.013
In order to obtain the highest expression level of excretory2secretory antigen Ts8B1 protein gene of Cys2 ticercus cellulosae in Escherichia coli,the factors affecting the protein expression were studied,including the medium,cul2 ture temperature,inducing time,the final concentration of inductor IPTG and Ampicillin (Amp),and the temperature change before and after induction. The results indicated that the expression of pGEX26P21/ Ts8B1 fusion protein reached the highest level when TB culture medium was used at 37 ℃for 3 h and then induction culture was carried out overnight at 32 ℃using 0. 2 mmol/L IPTGand 200 mmol/L Amp. The expression fusion protein accounted for 33 %of the total cell lysates. SDS2PAGE analysis showed that the molecular mass of pGEX26P21/ Ts8B1 fusion protein was about 33 kDa,the same as predicted. Western blot analysis indicated that the Cysticercus cellulose polyclonal antibody could be specifically bound to pGEX26P21/ Ts8B1 fusion protein. The results would be useful for obtaining the maximum yield of fusion protein and for researching its application value in detecting cysticercosis.
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