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.
WANG Qiu xia,, ZHANG Mei ying, ZHANG Gai ping, WANG Xuan nian,, NING Chang shen, LU Kun,, ZHANG Long xian, JIAN Fu chun
. Optimization of Prokaryotic Expression of Excretory-secretory Antigen Ts8B1 Protein Gene in Cysticercus cellulosae[J]. Acta Agriculturae Boreali-Sinica, 2009
, 24(3)
: 59
-63
.
DOI: 10.7668/hbnxb.2009.03.013
[1] 赵光辉,张改平,王选年,等.猪囊尾蚴18kD蛋白基因的克隆及其序列分析[J].河南农业科学,2006(12):87-90.
[2] 李海龙,景志忠,姜悦平,等.猪囊尾蚴124基因的克隆与表达[J].中国兽医科学,2006,36(07):547-551.
[3] Ferret E,Bonay P,Cuevas M F,et al.Molecular cloning and characterisation of Ts8B1,Ts8B2 and Ts8B3,three new mem-bers of the Taenia solium metacestede 8 kDa diagnostic anti-gen family[J].Mol Biechem Parasitol,2007,152:90-100.
[4] Assana E,Kanobana K,Tume C B,et al.Isolation of a 14 kDa antigen from Taenia solium cyst fluid by HPLC and its e-valuation in enzyme linked immunosorbent assay for diagnosis of porcine cysticercosis[J].Res Vet Sci,2007,82(3):370-376.
[5] Espindola N M,Vaz A J,Pardini A X,et al.Exeretory/secre-tory antigens(ES)from in-vitro cultures of Taenia crassiceps cystlcerci,and use ofan anti-ES monoclonal antibody for anti-gen detection in samples of cerebr-ospinal fluid from patients with neurocysticercosis[J].Ann Trop Med Parasitol,2002,96(4):361-368.
[6] Molinari J L,Garcy a-Mendoza E,De la Garza Y,et al.Dis-crimination between active and inactive neurocys-ticercosis by metacestode excretory/secretory antigens of Taenia solium in an enzymelinked immunosorbent assay[J].Am J Trop Med Hyg,2002,66(6):777-781.
[7] 王中全,崔晶.旋毛虫排泄分泌抗原的研究进展[J].中国预防兽医学报,2003,25(2):155-158.
[8] 孔建强,王伟,杜冠华,等.SARS冠状病毒未知功能小蛋白大肠杆菌表达及条件优化[J].药学学报,2007,2(9):1000-1006.
[9] 潘滨,吴建祥,李桂新,等.烟草曲茎病毒复制相关蛋白基因原核表达条件优化[J].浙江大学学报:农业与生命科学版,2007,33(1):24-28.
[10] 李艳,谭宏亮,何亚辉,等.大肠杆菌丙酮酸脱氢酶的克隆与表达优化[J].化学与生物工程,2007,24(10):28-31.
[11] 杨书慧,赵胜军,刘军,等.乳糖诱导甜蛋白Monellin在大肠杆菌中的表达[J].中国生物工程杂志,2008,28(3):53-58.
[12] Menzella H G,Ceccarelli E A,Gramajo H C.Novel Es-cherichia coli st rain allows efficient recombinant protein production using lactose as inducer[J].Biotech Bioeng,2003,82(7):809-817.
[13] 张毅,屈贤铭,杨胜利.乳糖作为诱导剂对重组目的蛋白表达的影响[J].生物工程学报,2000,16(4):464-468.
[14] Woyski D,Cupp-Vickery J R.Enhanced expression of cy-tochrome P450s from lac2 based plasmids using lactose as the inducer[J].Arch Biochem Biophys,2001,388(2):276-280.
[15] Monteiro R A,Souza E M,Yates M G,et al.Use of lactose to in duce expression of soluble NifA protein domains of Herbaspirillum seropedicae in Escherichia coli[J].Can J Microbiol,2000,46(11):1080-1090.
