生长抑素原核真核双表达质粒的构建与鉴定及作用机理研究
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摘要
原核真核双表达系统与单一的原核或真核表达系统相比,具有较大的优势,可以同时启动原核和真核双表达外源基因。以减毒猪霍乱沙门氏菌为运载菌的原核真核双表达系统,可以同时在细菌和真核细胞中表达同一蛋白质,且原核表达产物可以刺激机体免疫应答；同样,真核表达质粒在机体中的表达产物也可以刺激机体产生免疫应答反应。因此,理论上原核和真核双表达质粒可以刺激机体提高其免疫应答能力。
本研究应用基因克隆、细胞培养、RT-PCR、SDS-PAGE、Western blot和ELISA等技术,将原核启动子Ptrc片段克隆到非抗性筛选生长抑素真核表达质粒pGS/2SS-M4GFP-asd,构建生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd,分别转化asd-大肠杆菌χ6097和crp-/asd-双缺失减毒猪霍乱沙门氏菌C500,得到x6097 (pPtrc-S/2SS-M4GFP-asd)和C500 (pPtrc-S/2SS-M4GFP-asd)工程菌。接着对pPtrc-S/2SS-M4GFP-asd质粒和C500 (pPtrc-S/2SS-M4GFP-asd)工程菌进行原核、真核表达鉴定。将重组菌免疫4周龄昆明小鼠,检测小鼠血浆中抗SS抗体和抗C500水平,旨在探讨以减毒猪霍乱沙门氏菌为载体的生长抑素原核真核双表达质粒的免疫效力,为提高现有生长抑素基因疫苗的免疫效果,开发高效、安全的促生长疫苗奠定理论基础,并提供技术支撑。
1.生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd的构建与筛选
人工合成启动子Ptrc序列片段,将其亚克隆入pGS/2SS-M4GFP-asd真核表达质粒CMV启动子下游,成功构建了生长抑素原核真核双表达质粒(pPtrc-S/2SS-M4GFP-asd);分别转化大肠杆菌X6097和减毒猪霍乱沙门氏菌C500,经双酶切和测序鉴定结果表明,基因的插入位点、方向和序列完全正确,成功得到分别以大肠杆菌x6097 (pPtrc-S/2SS-M4GFP-asd)和减毒猪霍乱沙门氏菌C500 (pPtrc-S/2SS-M4GFP-asd)为载体的工程菌各1株。
2.生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd的表达与鉴定
将构建好的生长抑素工程菌C500 (pPtrc-S/2SS-M4GFP-asd)经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)和非IPTG诱导培养3h后,培养物中检出S/2SS-M4GFP融合蛋白,大小为55KDa；用Western blot方法鉴定证明,表达的融合蛋白可与抗生长抑素抗体结合；将生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd转染293FT细胞,48h后观察到绿色荧光,RT-PCR检测证实转录产物有S片段(352bp)、S/SS片段(491bp)和S/2SS(795bp),表明该质粒既可在原核细胞又可在真核细胞中进行表达。
3.生长抑素原核真核双表达重组菌免疫对小鼠免疫应答和生长的影响
将生长抑素原核真核双表达C500 (pPtrc-S/2SS-M4GFP-asd)工程菌分三个免疫剂量(高剂量0.2×1010CFU/只,中剂量0.2×109CFU/只,低剂量0.2×108CFU/只)免疫雌性昆明小鼠。在整个实验阶段,高剂量组增重分别比中剂量组、低剂量组和真核对照组(pGS/2SS-M4GFP-asd)提高5.5%、8.2%和7.1%,但组间差异不显著(P>0.05),且高剂量组料肉比最低(25.9：1)。初免后3w高剂量组产生了高于真核对照组6.7%的IgG抗体效价；加强免疫后高剂量组IgG抗体效价比真核对照组提高8.2%。初免后3w高剂量组比真核对照组IgG1抗体效价提高25%,但IgG1>IgG2a,表明免疫疫苗菌后机体倾向于产生Th2型免疫应答,以介导体液免疫反应为主。由此说明,生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd与生长抑素基因疫苗pGS/2SS-M4GFP-asd相比,能够引起更强的免疫应答反应尤其是体液免疫反应,从而更好地促进机体生长。
The prokaryotic-eukaryotic dual expression system compared to a single prokaryotic or eukaryotic expression system has greater advantages and can also start the prokaryotic and eukaryotic expression of foreign genes. The prokaryotic-eukaryotic dual expression system using attenuated strain of Salmonella choleraesuis as the carrier, could express the same proteins in bacteria and mammalian cells, and also can stimulate the immune response respectively by products of prokaryotic expression and eukaryotic expression.Therefore, in theory, prokaryotic-eukaryotic dual expression plasmid can stimulate the body's ability to improve their immune response.
To construct the prokaryotic-eukaryotic dual expression vector pPtrc-S/2SS-M4GFP-asd, gene cloning, cell culture, RT-PCR, SDS-PAGE, Western blot, ELISA and other technologies were applied in this research, and the original promoter Ptrc fragment was cloned into the somatostatin eukaryotic expression plasmid without resistence gene (pGS/2SS-M4GFP-asd), then the vector of pPtrc-S/2SS-M4GFP-asd was separately converted into E.colχs6097 and crp-/asd-double deletion attenuated Salmonella choleraesuis C500, soχ6097(pPtrc-S/2SS-M4GFP-asd) and C500 (pPtrc-S/2SS-M4GFP-asd) engineered strains were obtained. Then we studied the identification of pPtrc-S/2SS-M4GFP-asd plasmid and C500(pPtrc-S/2SS-M4GFP-asd) engineered bacteria on prokaryotic and eukaryotic expression. We used the recombinant bacteria to immune 4 week-age mice, then detected the levels of anti-SS antibody and anti-C500 antibody in plasma, in order to discuss the immune effect of attenuated Salmonella choleraesuis as the carrier of somatostatin prokaryotic-eukaryotic dual expression plasmid, and also to develop efficient and safe gene vaccine on growth for providing theoretical basis and technical support.
1. Construction and Screening of Somatostatin prokaryotic-eukaryotic expression plasmid pPtrc-S/2SS-M4GFP-asd
The prokaryotic promoter Ptrc was synthesized and subcloned into the CMV promoter downstream of the eukaryotic expression vector pGS/2SS-M4GFP-asd, the somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd was built, and also was converted into E.coliχ6097 and crp-/asd-double deletion attenuated Salmonella choleraesuis C500. It was proved that gene insertion site, direction and sequence were completely correct, so x6097(pPtrc-S/2SS-M4GFP-asd) and C500(pPtrc-S/2SS-M4GFP-asd) engineered strains were sucessfully obtained.
2. Expression and identification of somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd
The constructed somatostatin engineered strain C500(pPtrc-S/2SS-M4GFP-asd) were cultured for 3h, which were inducted by isopropyl-β-D-thio-pyran-galactoside (IPTG) and non IPTG, then the S/2SS-M4GFP fusion protein with the size of 55KDa was detected; using the method of Western blot, it was proved that the fusion protein could combined the anti-somatostatin antibody; when the somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd was transfected into 293FT cells, the green fluorescence was detected after 48h, and also RT-PCR transcription products were tested by S segment (352bp), S/SS fragment (491bp) and S/2SS(795bp), therefore the results showed that the plasmid could express respecticely in prokaryotic and eukaryotic cells.
3. Effects of the Prokaryotic-eukaryotic dual expression DNA immunization of somatostatin on growth and immune response in mice
The prokaryotic-eukaryotic dual expression of somatostatin C500 (pPtrc-S/2SS-M4GFP-asd) engineered bacteria was used to immune female kunming mice, by three immunization doses(high dose of 0.2×1010CFU/each, medium dose of 0.2×109CFU/each, low dose of 0.2×108CFU/each) to the immune female mice.Throughout the experimental stage, compared to medium-dose, low-dose and eukaryotic control group(pGS/2SS-M4GFP-asd), high-dose group had weight gain increased respectively by 5.5%,8.2%and 7.1%, without no significant difference between those groups (P>0.05), and also had the lowest feed conversion rate(25.9:1).3w after the first vaccination, high-dose group had a higher IgG antibody titer compared to eukaryotic control group by 6.7%; after strengthen vaccination, IgG antibody titers of high-dose group were higher than the eukaryotic control group by 8.2%.3w after the first vaccination, IgGl antibody titers of high-dose group were increased by the eukaryotic control group by 25%, but IgG1>IgG2a, so it was proved that after vaccination, the body tends to produce Th2 type immune response mainly mediate humoral immune response. Those showed that prokaryotic-eukaryotic dual expression plasmid of somatostatin DNA vaccine pGS/2SS-M4GFP-asd could cause stronger immune response especially humoral immune reaction, in order to enchance the relative growth compared with the somatostatin gene vaccine pGS/2SS-M4GFP-asd.
本研究应用基因克隆、细胞培养、RT-PCR、SDS-PAGE、Western blot和ELISA等技术,将原核启动子Ptrc片段克隆到非抗性筛选生长抑素真核表达质粒pGS/2SS-M4GFP-asd,构建生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd,分别转化asd-大肠杆菌χ6097和crp-/asd-双缺失减毒猪霍乱沙门氏菌C500,得到x6097 (pPtrc-S/2SS-M4GFP-asd)和C500 (pPtrc-S/2SS-M4GFP-asd)工程菌。接着对pPtrc-S/2SS-M4GFP-asd质粒和C500 (pPtrc-S/2SS-M4GFP-asd)工程菌进行原核、真核表达鉴定。将重组菌免疫4周龄昆明小鼠,检测小鼠血浆中抗SS抗体和抗C500水平,旨在探讨以减毒猪霍乱沙门氏菌为载体的生长抑素原核真核双表达质粒的免疫效力,为提高现有生长抑素基因疫苗的免疫效果,开发高效、安全的促生长疫苗奠定理论基础,并提供技术支撑。
1.生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd的构建与筛选
人工合成启动子Ptrc序列片段,将其亚克隆入pGS/2SS-M4GFP-asd真核表达质粒CMV启动子下游,成功构建了生长抑素原核真核双表达质粒(pPtrc-S/2SS-M4GFP-asd);分别转化大肠杆菌X6097和减毒猪霍乱沙门氏菌C500,经双酶切和测序鉴定结果表明,基因的插入位点、方向和序列完全正确,成功得到分别以大肠杆菌x6097 (pPtrc-S/2SS-M4GFP-asd)和减毒猪霍乱沙门氏菌C500 (pPtrc-S/2SS-M4GFP-asd)为载体的工程菌各1株。
2.生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd的表达与鉴定
将构建好的生长抑素工程菌C500 (pPtrc-S/2SS-M4GFP-asd)经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)和非IPTG诱导培养3h后,培养物中检出S/2SS-M4GFP融合蛋白,大小为55KDa；用Western blot方法鉴定证明,表达的融合蛋白可与抗生长抑素抗体结合；将生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd转染293FT细胞,48h后观察到绿色荧光,RT-PCR检测证实转录产物有S片段(352bp)、S/SS片段(491bp)和S/2SS(795bp),表明该质粒既可在原核细胞又可在真核细胞中进行表达。
3.生长抑素原核真核双表达重组菌免疫对小鼠免疫应答和生长的影响
将生长抑素原核真核双表达C500 (pPtrc-S/2SS-M4GFP-asd)工程菌分三个免疫剂量(高剂量0.2×1010CFU/只,中剂量0.2×109CFU/只,低剂量0.2×108CFU/只)免疫雌性昆明小鼠。在整个实验阶段,高剂量组增重分别比中剂量组、低剂量组和真核对照组(pGS/2SS-M4GFP-asd)提高5.5%、8.2%和7.1%,但组间差异不显著(P>0.05),且高剂量组料肉比最低(25.9：1)。初免后3w高剂量组产生了高于真核对照组6.7%的IgG抗体效价；加强免疫后高剂量组IgG抗体效价比真核对照组提高8.2%。初免后3w高剂量组比真核对照组IgG1抗体效价提高25%,但IgG1>IgG2a,表明免疫疫苗菌后机体倾向于产生Th2型免疫应答,以介导体液免疫反应为主。由此说明,生长抑素原核真核双表达质粒pPtrc-S/2SS-M4GFP-asd与生长抑素基因疫苗pGS/2SS-M4GFP-asd相比,能够引起更强的免疫应答反应尤其是体液免疫反应,从而更好地促进机体生长。
The prokaryotic-eukaryotic dual expression system compared to a single prokaryotic or eukaryotic expression system has greater advantages and can also start the prokaryotic and eukaryotic expression of foreign genes. The prokaryotic-eukaryotic dual expression system using attenuated strain of Salmonella choleraesuis as the carrier, could express the same proteins in bacteria and mammalian cells, and also can stimulate the immune response respectively by products of prokaryotic expression and eukaryotic expression.Therefore, in theory, prokaryotic-eukaryotic dual expression plasmid can stimulate the body's ability to improve their immune response.
To construct the prokaryotic-eukaryotic dual expression vector pPtrc-S/2SS-M4GFP-asd, gene cloning, cell culture, RT-PCR, SDS-PAGE, Western blot, ELISA and other technologies were applied in this research, and the original promoter Ptrc fragment was cloned into the somatostatin eukaryotic expression plasmid without resistence gene (pGS/2SS-M4GFP-asd), then the vector of pPtrc-S/2SS-M4GFP-asd was separately converted into E.colχs6097 and crp-/asd-double deletion attenuated Salmonella choleraesuis C500, soχ6097(pPtrc-S/2SS-M4GFP-asd) and C500 (pPtrc-S/2SS-M4GFP-asd) engineered strains were obtained. Then we studied the identification of pPtrc-S/2SS-M4GFP-asd plasmid and C500(pPtrc-S/2SS-M4GFP-asd) engineered bacteria on prokaryotic and eukaryotic expression. We used the recombinant bacteria to immune 4 week-age mice, then detected the levels of anti-SS antibody and anti-C500 antibody in plasma, in order to discuss the immune effect of attenuated Salmonella choleraesuis as the carrier of somatostatin prokaryotic-eukaryotic dual expression plasmid, and also to develop efficient and safe gene vaccine on growth for providing theoretical basis and technical support.
1. Construction and Screening of Somatostatin prokaryotic-eukaryotic expression plasmid pPtrc-S/2SS-M4GFP-asd
The prokaryotic promoter Ptrc was synthesized and subcloned into the CMV promoter downstream of the eukaryotic expression vector pGS/2SS-M4GFP-asd, the somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd was built, and also was converted into E.coliχ6097 and crp-/asd-double deletion attenuated Salmonella choleraesuis C500. It was proved that gene insertion site, direction and sequence were completely correct, so x6097(pPtrc-S/2SS-M4GFP-asd) and C500(pPtrc-S/2SS-M4GFP-asd) engineered strains were sucessfully obtained.
2. Expression and identification of somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd
The constructed somatostatin engineered strain C500(pPtrc-S/2SS-M4GFP-asd) were cultured for 3h, which were inducted by isopropyl-β-D-thio-pyran-galactoside (IPTG) and non IPTG, then the S/2SS-M4GFP fusion protein with the size of 55KDa was detected; using the method of Western blot, it was proved that the fusion protein could combined the anti-somatostatin antibody; when the somatostatin prokaryotic-eukaryotic dual expression plasmid pPtrc-S/2SS-M4GFP-asd was transfected into 293FT cells, the green fluorescence was detected after 48h, and also RT-PCR transcription products were tested by S segment (352bp), S/SS fragment (491bp) and S/2SS(795bp), therefore the results showed that the plasmid could express respecticely in prokaryotic and eukaryotic cells.
3. Effects of the Prokaryotic-eukaryotic dual expression DNA immunization of somatostatin on growth and immune response in mice
The prokaryotic-eukaryotic dual expression of somatostatin C500 (pPtrc-S/2SS-M4GFP-asd) engineered bacteria was used to immune female kunming mice, by three immunization doses(high dose of 0.2×1010CFU/each, medium dose of 0.2×109CFU/each, low dose of 0.2×108CFU/each) to the immune female mice.Throughout the experimental stage, compared to medium-dose, low-dose and eukaryotic control group(pGS/2SS-M4GFP-asd), high-dose group had weight gain increased respectively by 5.5%,8.2%and 7.1%, without no significant difference between those groups (P>0.05), and also had the lowest feed conversion rate(25.9:1).3w after the first vaccination, high-dose group had a higher IgG antibody titer compared to eukaryotic control group by 6.7%; after strengthen vaccination, IgG antibody titers of high-dose group were higher than the eukaryotic control group by 8.2%.3w after the first vaccination, IgGl antibody titers of high-dose group were increased by the eukaryotic control group by 25%, but IgG1>IgG2a, so it was proved that after vaccination, the body tends to produce Th2 type immune response mainly mediate humoral immune response. Those showed that prokaryotic-eukaryotic dual expression plasmid of somatostatin DNA vaccine pGS/2SS-M4GFP-asd could cause stronger immune response especially humoral immune reaction, in order to enchance the relative growth compared with the somatostatin gene vaccine pGS/2SS-M4GFP-asd.
引文
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