IL-6与GM-CSF基因融合表达生长抑素基因疫苗对小鼠生长的影响
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摘要
白细胞介素-6(IL-6)是常用的免疫增强剂。为了探讨其在基因免疫中的免疫增强作用,本研究应用基因克隆、细胞培养、酶联免疫测定、RT-PCR等技术,首先克隆了牛的IL-6基因,在生长抑素DNA疫苗pGS/2SS-asd基础上插入牛的IL-6基因,构建非抗性筛选生长抑素真核表达质粒pGS/2SS-IL6-asd,并电转化至缺失asd基因的减毒猪霍乱沙门氏菌,将重组菌肌注免疫小鼠后,旨在：①探讨IL-6和GM-CSF融合表达对生长抑素DNA疫苗免疫反应和增重效果的影响；②评估其安全性,为开发安全、高效的促生长DNA疫苗,加快其临床应用奠定基础。
1.牛IL-6基因克隆和序列分析
用ConA、PHA体外刺激诱导牛外周血单核细胞(PBMC),提取单核细胞总RNA后,利用RT-PCR克隆牛IL-6基因。序列分析表明,牛IL-6基因cDNA开放阅读框为624bp,与Genbank数据库中登载的牛IL-6序列(序列号：X57317.1)进行序列比较,结果有99.84%的同源性。将牛的IL-6基因插入T载体(pEASY-T1-simple)保存。
2.非抗性筛选生长抑素真核表达质粒pGS/2SS-IL6-asd的构建与鉴定
将牛IL-6基因插入到pGS/2SS-asd质粒的下游,构建带有IL-6和GM-CSF融合表达的非抗性筛选生长抑素真核表达质粒pGS/2SS-IL6-asd。酶切、测序鉴定结果表明,基因的插入位点、方向、序列完全正确。RT-PCR结果表明,pGS/2SS-IL6-asd质粒转染细胞48h后能检测到转录产物。
3.非抗性筛选生长抑素DNA疫苗pGS/2SS-IL6-asd免疫小鼠的免疫反应和促生长效果
将所构建的质粒pGS/2SS-asd电转化至缺失asd基因的减毒猪霍乱沙门氏菌C500,获得非抗性筛选生长抑素DNA疫苗C500(pGS/2SS-asd)。将50只雌性昆明小鼠随机分为6组,A、B、C三组分别用3种剂量(1×1010CFU／只,1×109CFU／只,1×108CFU／只)pGS/2SS-IL6-asd疫苗进行免疫；D组免疫不带IL-6的C500基因疫苗,即pGS/2SS-asd,剂量为109cfu/mL;E组免疫沙门氏菌空菌C500(109cfu/mL),F组免疫PBS。各组免疫方式均为肌肉注射,体积为0.2mL。在首次免疫后,第2周,用同等剂量的疫苗或对照液进行加强免疫一次。并分别在0(初次免疫前)、14(二免前)、28、42和56天同一时段采血。ELISA结果显示,A、B、C、D组小鼠均可以检测到抗SS抗体,且随免疫时间的延长,呈现升高的趋势。pGS/2SS-IL6-asd高剂量组和中剂量组取得了较高的抗体水平,极显著高于低剂量组和(?)pGS/2SS-asd组(p<0.01)。每周称量小鼠体重,发现pGS/2SS-IL6-asd高剂量组增重效果明显高于低剂量组和(?)pGS/2SS-asd组。综上所述,IL-6和GM-CSF融合表达对生长抑素DNA疫苗有一定的免疫增强作用,pGS/2SS-IL6-asd效果要优于pGS/2SS-asd。4.染色体整合分析
将试验末期雌性昆明小鼠处死后,采集心、肝、脾、肺、肾、卵巢和肌肉组织,提取基因组DNA,PCR法检测质粒DNA的染色体整合情况。结果表明,在PCR的最大灵敏度(10拷贝/μg基因组)范围之内,没有发现质粒整合至基因组,提示即使发生整合突变,其发生频率也低于自发突变至少两个数量级。
IL-6 gene was commonly used as immunopotentiators. In ord to discusse its immunologic enhancement at genetic immunization, a series of techniques were used in this study, such as gene cloning, cell culture, ELISA, RT-PCR. IL-6 gene of cattle was cloned in order to construct somatostatin eukaryotic expression plasmid pGS/2SS-IL6-asd without antibiotic resistance gene, on the basis of somatostatin DNA vaccine pGS/2SS-asd. Then, the plasmid pGS/2SS-IL6-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain (with deletion of asd genes) by electroporation, and the recombinant strain C500(pGS/2SS-IL6-asd) was intra-muscularly immunized against mice. The immunogenicity of fusion-expression plasmid (pGS/2SS-IL6-asd) harboring GM-CSF and IL-6 gene, and safety assessment were discussed here to develop gene immunization techniques to promote growth of animals and to accelerate clinical application of SS gene vaccines.
1. Cloning and sequencing of IL-6 gene
IL-6 gene of cattle was cloned by RT-PCR. The RNA of cells were stimulated and induced by ConA and PHA. cDNA encoding cattle IL-6 was cloned and sequence analysis showed that it was 624 by in length. Compared with the sequence of IL-6 gene of cattle publish in Genbank (X57317.1).Homology of the cattle IL-6 cloned coding sequence was 99.84%.Cattle IL-6 gene was cloned into pEASY-T1-simple to store.
2. Construction and identification of somatostatin eukaryotic expression plasmid pGS/2SS-IL6-asd without antibiotic resistance gene
The IL-6 gene was then fused into 3'end of GS/2SS gene in the proper reading frame to construct fusion-expression plasmid pGS/2SS-IL6-asd harboring GM-CSF and IL-6 gene without antibiotic resistance gene. The insertion site, direction and sequence of the genes were identified to be correct by restriction endonuclease digestion and sequencing. RT-PCR result showed that the GS/2SS-IL6 somatostatin gene could be detected.
3. The somatostatin DNA vaccine pGS/2SS-IL6-asd without antibiotic resistance gene influence the growth of mice
The plasmid pGS/2SS-IL6-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain by electroporation, and formed SS DNA vaccine C500(pGS/2SS-IL6-asd). A total of 50 female Kungming mice were divided into 6 groups.Group A, B and C were immunized with pGS/2SS-IL6-asd by three different dose(1×1010CFU per mice,1×109CFU per mice,1×108 CFU per mice). Group D was immunized with pGS/2SS-asd(1×109CFU per mice). Group E was immunized with C500(1×109CFU per mice). Group F was immunized with PBS. Enhance immunization after two weeks from the first immunization. Blood collection was at 0,14,28,42 and 56 days. Results of ELISA showed that the anti-SS-antibodies were detected in the A to D groups, and the titers of antibodies presented a tendency of increase along with the extension of immunization. The high and middle dose group immunized with pGS/2SS-IL6-asd were received the high antibodies compared with the low dose group and pGS/2SS-asd group (p<0.01).Every week weight the body weight of mice. For the mean body weight of mice, the high dose group immunized with pGS/2SS-IL6-asd was also obtained the better body gain than the other groups. In conclusion, fusion-expression of IL-6 and GM-CSF can stimulate immune responses and enhance spleen lymphocyte proliferation responses of immunized mice to specific antigen. Immune response of fusion-expression plasmid of pGS/2SS-IL6-asd is superior to pGS/2SS-asd.
4. Chromosomal integration analysis
At the termination of Kungming mice, the mice were killed to collect the tissues of heart, liver, spleen, lung, kidney, muscle and ovary. The genomic DNA was extracted, and then the genomic DNA samples were analyzed by sensitive PCR method. There was no evidence of integration to sensitivity of about 10 copy/μg DNA. If integration occurred at all, the frequency would be at least two orders of magnitude below the spontaneous mutation rate.
1.牛IL-6基因克隆和序列分析
用ConA、PHA体外刺激诱导牛外周血单核细胞(PBMC),提取单核细胞总RNA后,利用RT-PCR克隆牛IL-6基因。序列分析表明,牛IL-6基因cDNA开放阅读框为624bp,与Genbank数据库中登载的牛IL-6序列(序列号：X57317.1)进行序列比较,结果有99.84%的同源性。将牛的IL-6基因插入T载体(pEASY-T1-simple)保存。
2.非抗性筛选生长抑素真核表达质粒pGS/2SS-IL6-asd的构建与鉴定
将牛IL-6基因插入到pGS/2SS-asd质粒的下游,构建带有IL-6和GM-CSF融合表达的非抗性筛选生长抑素真核表达质粒pGS/2SS-IL6-asd。酶切、测序鉴定结果表明,基因的插入位点、方向、序列完全正确。RT-PCR结果表明,pGS/2SS-IL6-asd质粒转染细胞48h后能检测到转录产物。
3.非抗性筛选生长抑素DNA疫苗pGS/2SS-IL6-asd免疫小鼠的免疫反应和促生长效果
将所构建的质粒pGS/2SS-asd电转化至缺失asd基因的减毒猪霍乱沙门氏菌C500,获得非抗性筛选生长抑素DNA疫苗C500(pGS/2SS-asd)。将50只雌性昆明小鼠随机分为6组,A、B、C三组分别用3种剂量(1×1010CFU／只,1×109CFU／只,1×108CFU／只)pGS/2SS-IL6-asd疫苗进行免疫；D组免疫不带IL-6的C500基因疫苗,即pGS/2SS-asd,剂量为109cfu/mL;E组免疫沙门氏菌空菌C500(109cfu/mL),F组免疫PBS。各组免疫方式均为肌肉注射,体积为0.2mL。在首次免疫后,第2周,用同等剂量的疫苗或对照液进行加强免疫一次。并分别在0(初次免疫前)、14(二免前)、28、42和56天同一时段采血。ELISA结果显示,A、B、C、D组小鼠均可以检测到抗SS抗体,且随免疫时间的延长,呈现升高的趋势。pGS/2SS-IL6-asd高剂量组和中剂量组取得了较高的抗体水平,极显著高于低剂量组和(?)pGS/2SS-asd组(p<0.01)。每周称量小鼠体重,发现pGS/2SS-IL6-asd高剂量组增重效果明显高于低剂量组和(?)pGS/2SS-asd组。综上所述,IL-6和GM-CSF融合表达对生长抑素DNA疫苗有一定的免疫增强作用,pGS/2SS-IL6-asd效果要优于pGS/2SS-asd。4.染色体整合分析
将试验末期雌性昆明小鼠处死后,采集心、肝、脾、肺、肾、卵巢和肌肉组织,提取基因组DNA,PCR法检测质粒DNA的染色体整合情况。结果表明,在PCR的最大灵敏度(10拷贝/μg基因组)范围之内,没有发现质粒整合至基因组,提示即使发生整合突变,其发生频率也低于自发突变至少两个数量级。
IL-6 gene was commonly used as immunopotentiators. In ord to discusse its immunologic enhancement at genetic immunization, a series of techniques were used in this study, such as gene cloning, cell culture, ELISA, RT-PCR. IL-6 gene of cattle was cloned in order to construct somatostatin eukaryotic expression plasmid pGS/2SS-IL6-asd without antibiotic resistance gene, on the basis of somatostatin DNA vaccine pGS/2SS-asd. Then, the plasmid pGS/2SS-IL6-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain (with deletion of asd genes) by electroporation, and the recombinant strain C500(pGS/2SS-IL6-asd) was intra-muscularly immunized against mice. The immunogenicity of fusion-expression plasmid (pGS/2SS-IL6-asd) harboring GM-CSF and IL-6 gene, and safety assessment were discussed here to develop gene immunization techniques to promote growth of animals and to accelerate clinical application of SS gene vaccines.
1. Cloning and sequencing of IL-6 gene
IL-6 gene of cattle was cloned by RT-PCR. The RNA of cells were stimulated and induced by ConA and PHA. cDNA encoding cattle IL-6 was cloned and sequence analysis showed that it was 624 by in length. Compared with the sequence of IL-6 gene of cattle publish in Genbank (X57317.1).Homology of the cattle IL-6 cloned coding sequence was 99.84%.Cattle IL-6 gene was cloned into pEASY-T1-simple to store.
2. Construction and identification of somatostatin eukaryotic expression plasmid pGS/2SS-IL6-asd without antibiotic resistance gene
The IL-6 gene was then fused into 3'end of GS/2SS gene in the proper reading frame to construct fusion-expression plasmid pGS/2SS-IL6-asd harboring GM-CSF and IL-6 gene without antibiotic resistance gene. The insertion site, direction and sequence of the genes were identified to be correct by restriction endonuclease digestion and sequencing. RT-PCR result showed that the GS/2SS-IL6 somatostatin gene could be detected.
3. The somatostatin DNA vaccine pGS/2SS-IL6-asd without antibiotic resistance gene influence the growth of mice
The plasmid pGS/2SS-IL6-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain by electroporation, and formed SS DNA vaccine C500(pGS/2SS-IL6-asd). A total of 50 female Kungming mice were divided into 6 groups.Group A, B and C were immunized with pGS/2SS-IL6-asd by three different dose(1×1010CFU per mice,1×109CFU per mice,1×108 CFU per mice). Group D was immunized with pGS/2SS-asd(1×109CFU per mice). Group E was immunized with C500(1×109CFU per mice). Group F was immunized with PBS. Enhance immunization after two weeks from the first immunization. Blood collection was at 0,14,28,42 and 56 days. Results of ELISA showed that the anti-SS-antibodies were detected in the A to D groups, and the titers of antibodies presented a tendency of increase along with the extension of immunization. The high and middle dose group immunized with pGS/2SS-IL6-asd were received the high antibodies compared with the low dose group and pGS/2SS-asd group (p<0.01).Every week weight the body weight of mice. For the mean body weight of mice, the high dose group immunized with pGS/2SS-IL6-asd was also obtained the better body gain than the other groups. In conclusion, fusion-expression of IL-6 and GM-CSF can stimulate immune responses and enhance spleen lymphocyte proliferation responses of immunized mice to specific antigen. Immune response of fusion-expression plasmid of pGS/2SS-IL6-asd is superior to pGS/2SS-asd.
4. Chromosomal integration analysis
At the termination of Kungming mice, the mice were killed to collect the tissues of heart, liver, spleen, lung, kidney, muscle and ovary. The genomic DNA was extracted, and then the genomic DNA samples were analyzed by sensitive PCR method. There was no evidence of integration to sensitivity of about 10 copy/μg DNA. If integration occurred at all, the frequency would be at least two orders of magnitude below the spontaneous mutation rate.
引文
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