大肠杆菌热敏性肠毒素双突变体构建及佐剂功能研究
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摘要
产肠毒素大肠杆菌产生的热敏性肠毒素(Heat-labile enterotoxin,LT)具有粘膜免疫原性和免疫佐剂功能,但具有很强的毒性,只有降低或去除LT蛋白的毒性,才能有效地利用其佐剂功能。为了得到安全性好的LT蛋白,选择其毒性关键位点63、72和192进行双突变研究,以期获得无毒但具有免疫佐剂功能的热敏性肠毒素。
以pET30a-LTK63、pET30a-LTR72质粒为模板,利用重叠延伸PCR扩增技术体外获得LT双突变体LTK63/G192、LTR72/G192基因,双酶切处理双突变体和pET30a载体,构建了pET30a-LTK63/G192、pET30a-LTR72/G192表达载体,PCR和酶切鉴定出阳性质粒进行测序,结果表明构建的表达载体阅读框架正确,突变体在192位点引入甘氨酸密码子(AGA→GGA)。重组质粒转化BL21(DE3)菌株,IPTG诱导,诱导表达产物用SDS-PAGE检测,各菌株均表达出约33kDa和13kDa的两个蛋白带,与LTA、LTB亚基分子量相吻合。
Western-blot检测,诱导表达的33KD、13KD蛋白均可与抗His抗体发生特异性免疫反应。表达蛋白用Ni-NTA Resin进行纯化,胰酶处理后,以DEABAG为底物,检测重组蛋白的ADP-核糖转移酶活性。结果,重组蛋白酶活性均显著低于野生型蛋白,而与阴性对照PBS相近。Patent-mouse毒性试验检测显示,LTK63/G192,LTR72/G192双突变体蛋白毒性均有不同程度降低,二者与LT相比差异均极显著,与PBS差异显著,且LTR72/G192的毒性略高于LTK63/G192。
LTK63/G192、LTR72/G192、LT蛋白分别与鸡新城疫低毒力活疫苗(NDV)一起经滴鼻免疫小鼠、雏鸡。经间接ELISA检测小鼠血清IgG抗体、鼻液IgA抗体水平。结果,NDV与LT双突变体蛋白免疫小鼠、雏鸡的血清IgG、局部粘膜IgA抗体水平均高于单独使用NDV免疫组,且LTR72/G192组抗体水平高于LTK63/G192组。经MTT法分析鸡脾脏T淋巴细胞增殖反应,结果发现,LT及LT双突变体蛋白免疫组的T淋巴细胞增殖反应均高于单独使用NDV免疫组及PBS空白对照但,但LTR72/G192与NDV免疫组T淋巴细胞增殖能力最强。
研究获得了减毒LT双突变体LTR72/G192、LTK63/G192表达载体,并证实其表达产物ADP-核糖转移酶活性和Patent-mouse毒性均低于LT野生性蛋白,且均具有良好粘膜佐剂活性,为临床开发其利用粘膜免疫佐剂功能奠定了基础。
Escherichia coli heat-labile enterotoxin (LT) generated from enterotoxigenic Escherichia coli (ETEC) is known to be a potent mucosal immunogen and immunoadjuvant towards co-administered antigens, but LT is toxic. toxicity must be declined or removed in order to utilize the adjuvant activity,. The key sites related to toxicity 63, 72, 192 were selected for double-mutants. The bioactivities of the double-mutants were tested and layed foundation for further adjuvant.
LTK63/G192 and LTR72/G192 were made by gene SOEing PCR using pET30aLTK63、pET30a-LTR72 as template. Destination genes and pET30a were excised by NcoI and SalI and then inserted into pET30a. Recombinant plasmids were transformed into E. coli DH5αrespectively. The positive recombinant plasmids LTK63/G192 and LTR72/G192 were selected by PCR, restriction analysis and nucleotide sequencing which indicated the replacement AGA (192th) with GGA. The positive plasmids were transformed into the host E. coli BL21(DE3). E.coli BL21 (DE3) was induced to express by IPTG.The expressed proteins were identified by SDS- PAGE and Western-blot using Anti-His tag antibody.
protein subunits LTA and LTB reacted with Anti-His tag antibody in Western-blot detection, The results showed that the two protein subunits were expressed successfully and the expressed protiens have the same immune activity as wtLT protein. The proteins were purified by Ni-NTA resin. The ADP-ribosyltransferase activity of wtLT and mutants treated with trypsin was determined using p-diethylamino-(benzylidinea-mino)-guanidine (DEABAG) as substrate. ADP-ribosyltransferase activity of the mutants was less than wtLT and approaches in the negative control group, LTK63/G192, LTR72/G192 double-mutant enzyme activity is also close. The toxicity assay of Patent-mouse showed that LTK63/G192,LTR72/G192 toxicity were all significantly reduced, LTR72/G192 toxicity is higher than LTK63/G192. LTK63/G192, LTR72/G192 and LT compare the difference to be the most significant. LTK63/G192, LTR72/G192 and PBS are more significant.than the difference.
The ability of LTK63/G192,LTR72/G192,LT to act as a mucosal adjuvant was tested by immunizing mouse and chickens intranasally (i.n.), using Newcastle vaccine for chickens as a bystander antigen.That results showed that mucosal adjuvanticity of LTR72/G192 were most significant. Proliferation of spleen T cells of chickens induced by NDV, test showed: LTR72/ G192 + NDV is the most effective in proliferation react.Thus double-mutants of heat-labile enterotoxin can act as effective intranasal mucosal adjuvants.
以pET30a-LTK63、pET30a-LTR72质粒为模板,利用重叠延伸PCR扩增技术体外获得LT双突变体LTK63/G192、LTR72/G192基因,双酶切处理双突变体和pET30a载体,构建了pET30a-LTK63/G192、pET30a-LTR72/G192表达载体,PCR和酶切鉴定出阳性质粒进行测序,结果表明构建的表达载体阅读框架正确,突变体在192位点引入甘氨酸密码子(AGA→GGA)。重组质粒转化BL21(DE3)菌株,IPTG诱导,诱导表达产物用SDS-PAGE检测,各菌株均表达出约33kDa和13kDa的两个蛋白带,与LTA、LTB亚基分子量相吻合。
Western-blot检测,诱导表达的33KD、13KD蛋白均可与抗His抗体发生特异性免疫反应。表达蛋白用Ni-NTA Resin进行纯化,胰酶处理后,以DEABAG为底物,检测重组蛋白的ADP-核糖转移酶活性。结果,重组蛋白酶活性均显著低于野生型蛋白,而与阴性对照PBS相近。Patent-mouse毒性试验检测显示,LTK63/G192,LTR72/G192双突变体蛋白毒性均有不同程度降低,二者与LT相比差异均极显著,与PBS差异显著,且LTR72/G192的毒性略高于LTK63/G192。
LTK63/G192、LTR72/G192、LT蛋白分别与鸡新城疫低毒力活疫苗(NDV)一起经滴鼻免疫小鼠、雏鸡。经间接ELISA检测小鼠血清IgG抗体、鼻液IgA抗体水平。结果,NDV与LT双突变体蛋白免疫小鼠、雏鸡的血清IgG、局部粘膜IgA抗体水平均高于单独使用NDV免疫组,且LTR72/G192组抗体水平高于LTK63/G192组。经MTT法分析鸡脾脏T淋巴细胞增殖反应,结果发现,LT及LT双突变体蛋白免疫组的T淋巴细胞增殖反应均高于单独使用NDV免疫组及PBS空白对照但,但LTR72/G192与NDV免疫组T淋巴细胞增殖能力最强。
研究获得了减毒LT双突变体LTR72/G192、LTK63/G192表达载体,并证实其表达产物ADP-核糖转移酶活性和Patent-mouse毒性均低于LT野生性蛋白,且均具有良好粘膜佐剂活性,为临床开发其利用粘膜免疫佐剂功能奠定了基础。
Escherichia coli heat-labile enterotoxin (LT) generated from enterotoxigenic Escherichia coli (ETEC) is known to be a potent mucosal immunogen and immunoadjuvant towards co-administered antigens, but LT is toxic. toxicity must be declined or removed in order to utilize the adjuvant activity,. The key sites related to toxicity 63, 72, 192 were selected for double-mutants. The bioactivities of the double-mutants were tested and layed foundation for further adjuvant.
LTK63/G192 and LTR72/G192 were made by gene SOEing PCR using pET30aLTK63、pET30a-LTR72 as template. Destination genes and pET30a were excised by NcoI and SalI and then inserted into pET30a. Recombinant plasmids were transformed into E. coli DH5αrespectively. The positive recombinant plasmids LTK63/G192 and LTR72/G192 were selected by PCR, restriction analysis and nucleotide sequencing which indicated the replacement AGA (192th) with GGA. The positive plasmids were transformed into the host E. coli BL21(DE3). E.coli BL21 (DE3) was induced to express by IPTG.The expressed proteins were identified by SDS- PAGE and Western-blot using Anti-His tag antibody.
protein subunits LTA and LTB reacted with Anti-His tag antibody in Western-blot detection, The results showed that the two protein subunits were expressed successfully and the expressed protiens have the same immune activity as wtLT protein. The proteins were purified by Ni-NTA resin. The ADP-ribosyltransferase activity of wtLT and mutants treated with trypsin was determined using p-diethylamino-(benzylidinea-mino)-guanidine (DEABAG) as substrate. ADP-ribosyltransferase activity of the mutants was less than wtLT and approaches in the negative control group, LTK63/G192, LTR72/G192 double-mutant enzyme activity is also close. The toxicity assay of Patent-mouse showed that LTK63/G192,LTR72/G192 toxicity were all significantly reduced, LTR72/G192 toxicity is higher than LTK63/G192. LTK63/G192, LTR72/G192 and LT compare the difference to be the most significant. LTK63/G192, LTR72/G192 and PBS are more significant.than the difference.
The ability of LTK63/G192,LTR72/G192,LT to act as a mucosal adjuvant was tested by immunizing mouse and chickens intranasally (i.n.), using Newcastle vaccine for chickens as a bystander antigen.That results showed that mucosal adjuvanticity of LTR72/G192 were most significant. Proliferation of spleen T cells of chickens induced by NDV, test showed: LTR72/ G192 + NDV is the most effective in proliferation react.Thus double-mutants of heat-labile enterotoxin can act as effective intranasal mucosal adjuvants.
引文
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