新型纳米抗龋DNA疫苗的制备及免疫学效应研究

发布时间：2018-06-23 00:08

本文选题：龋齿 + DNA疫苗　；参考：《第二军医大学》2014年硕士论文

【摘要】：近年来，随着人们生活质量的提高和老龄化程度的加深，越来越多的人开始关注口腔卫生。龋齿的发病率仅次于流感。轻度的龋齿影响咀嚼功能，重度龋齿可以引起根尖周病、牙髓病、颌骨炎症等并发症，严重影响人类全身健康和生存质量。由于我国人口众多、医疗资源有限，研制易于普及的龋病疫苗以防治龋病、降低龋病发病率对于保障国民健康显得尤为重要。变形链球菌（S. mutants）是公认的主要致龋因素之一，wapA蛋白是变形链球菌的表面蛋白，参与变形链球菌在宿主牙齿上的聚集粘附和菌斑形成。本文第一部分把变形链球菌UA159的编码wapA蛋白的全长基因序列重组到真核表达载体pVAX1上构建抗龋DNA疫苗pVAX1-wapA，建立其原核大肠杆菌（E.coli）表达系统。通过酶切与测序比对鉴定，构建的质粒序列正确，无移码，突变，碱基缺失等现象。并提取纯化了大量的pVAX1-wapA以用于后续的处方设计和体内外评价实验。本文第二部分构建了wapA蛋白的原核大肠杆菌表达系统，对时间，温度以及IPTG浓度等表达条件进行优化，结果显示最佳诱导表达条件是0.1mM IPTG，37℃，5h。通过亲和凝胶过滤的层析方法纯化得到了5.2mg，纯度为90%的wapA重组蛋白。本文第三部分实验以甲基丙烯酰氧乙基三甲基氯化铵（TMAEMC）修饰壳聚糖（CS），通过自由基聚合反应合成了水溶性良好的季铵化壳聚糖(CSTM)。通过1H-NMR和FT-IR对其结构进行了确证。合成的季铵化壳聚糖季铵化程度为52%。建立了质粒定量的荧光分光光度法，最佳发射波长464nm，最佳激发波长350nm，狭缝宽度5nm，质粒浓度0.07-1.75μg/ml范围内线性良好，回收率在97.6-99.9%，精密度高。并以壳聚糖及其衍生物季铵化壳聚糖为载体材料制备空白纳米粒，对纳米粒形成的关键因素：壳聚糖溶液浓度，pH值，N/P，TPP浓度，以纳米粒的粒径和zeta电位为指标进行考察。基于上述结论，对包载DNA的壳聚糖纳米粒的处方条件进行优化，最终确定处方条件为：CS溶液浓度为0.25mg/ml，缓冲体系pH为5.5，TPP浓度溶液0.02mM，pVAX1-wapA150ng/μl。CSTM溶液浓度为5mg/ml，缓冲体系pH为5.0，TPP浓度溶液0.02mM，pVAX1-wapA300ng/μl。制备了包载DNA疫苗粒径均一，形态圆整的纳米粒。CSTM纳米粒粒径为222.5nm，Zeta电位为19.6mV，包封率为87.66%，载药量为4.96%。制备的CS纳米粒粒径为219.2nm，Zeta电位为24.7mV，，包封率为91.24%，载药量为34.22%。在此基础上，本文第四部分实验使用包载质粒的纳米粒进行细胞转染，通过Realtime-PCR检测细胞对质粒编码wapA基因的转录表达水平。结果显示裸质粒pVAX1-wapA直接转染的细胞几乎检测不到wapA基因的mRNA转录。以与lipo2000共转染的pVAX1-wapA的转录水平为1，季铵化壳聚糖纳米粒包载的pVAX1-wapA转染细胞后wapA基因的mRNA水平为0.87，是壳聚糖的纳米粒包载的pVAX1-wapA转录水平的3.18倍。体外细胞转染结果表明，壳聚糖纳米粒和季铵化壳聚糖纳米粒均可以促进pVAX1-wapA进入细胞，并成功被转录。而裸质粒几乎无法进入细胞进行正常的转录翻译。本文第五部分实验首先使用前期构建的DNA疫苗和给药系统免疫正常小鼠，采用ELISA检测小鼠的唾液IgA和血清IgG水平。结果发现，DNA疫苗通过肌注途径可以成功激发小鼠免疫系统，产生IgG抗体水平的升高，证明了构建的重组质粒pVAX1-wapA具有免疫原性。并且壳聚糖纳米粒递送系统可以提高疫苗肌注的免疫原性，但肌注免疫途径并不能诱导粘膜免疫，实验结果没有观察到IgA抗体水平的显著升高。建立了ELISA法用于测定抗wapA蛋白特异性IgG和IgA抗体，方法批内和批间的变异系数均小于10%，重现性好。为了进一步证实我们构建的DNA疫苗的抗龋效果和能否有效激发粘膜免疫，采用致龋菌定植和致龋饲料建立定菌大鼠龋齿模型，增加了鼻腔免疫途径，对前期构建的新型抗龋齿疫苗对龋齿的预防和治疗作用进行评价。动物模型龋损评分结果显示，鼻腔免疫DNA疫苗纳米粒组龋损程度最低，该组的唾液IgA和血清IgG抗体水平也显著提高。肌注DNA疫苗和包载DNA疫苗的纳米粒组龋损也有降低，但评分都要高于包载DNA疫苗的纳米粒的鼻腔免疫组，肌注DNA疫苗和包载DNA疫苗虽然引起了高水平的血清IgG，但引起的唾液IgA水平都要低于鼻腔免疫组。本部分结果说明季铵化壳聚糖纳米粒具有良好的鼻腔免疫效果，适合作为鼻腔免疫的载体。综上所述，本文针对一种新的抗龋抗原wapA，构建了一种全新的重组质粒pVAX1-wapA用于防龋，并揭示wapA蛋白可以作为一个抗龋靶点。利用壳聚糖纳米粒无毒、安全、有效的穿细胞能力，提高了DNA疫苗的免疫效率。利用壳聚糖可促进DNA粘膜粘附和吸收，同时具有免疫佐剂的作用。针对龋齿疫苗采用鼻腔接种，提高DNA疫苗黏膜免疫效果，相比于肌注免疫显示更强的抗龋活性。包载pVAX1-wapA的CSTM纳米粒作为鼻腔免疫疫苗具有高效、安全的特点，显示出良好的应用潜力。
[Abstract]:In recent years, with the improvement of people's quality of life and the deepening of aging, more and more people begin to pay attention to oral hygiene. The incidence of dental caries is second only to influenza. Mild caries affect masticatory function. Severe caries can cause periapical disease, dental pulp disease, jaw inflammation and other complications, which seriously affect the whole body health and quality of life. Because of the large population and limited medical resources, the development of dental caries vaccine is easy to popularize to prevent caries, and it is very important to reduce the incidence of caries to protect the national health.
Streptococcus mutans (S. mutants) is recognized as one of the main caries inducing factors. WapA protein is the surface protein of Streptococcus mutans, and is involved in the aggregation and adhesion and plaque formation of Streptococcus mutans on the host teeth.
In the first part of this paper, the whole long gene sequence of the encoded wapA protein of Streptococcus mutans UA159 was reorganized into the eukaryotic expression vector pVAX1 to construct the anti caries DNA vaccine pVAX1-wapA, and the expression system of its prokaryotic Escherichia coli (E.coli) was established. A large number of pVAX1-wapA were extracted and purified for subsequent formulation design and in vivo and in vitro evaluation experiments.
In the second part of this paper, the expression system of prokaryotic Escherichia coli of wapA protein was constructed and the expression conditions of time, temperature and IPTG concentration were optimized. The results showed that the best expression condition was 0.1mM IPTG, 37 C, and 5h. was purified by affinity gel filtration chromatography to obtain the wapA recombinant protein with the purity of 90%.
In the third part of this third part, a water-soluble quaternized chitosan (CSTM) was synthesized by the free radical polymerization of chitosan (CS) modified with methacryl ethoxy ethyl three methyl ammonium chloride (TMAEMC). The structure of the chitosan was confirmed by 1H-NMR and FT-IR. The quaternizing degree of the synthesized Ji An chitosan was 52%. and the plasmid quantitative was established. Fluorescence spectrophotometry, the optimum emission wavelength 464nm, the optimum excitation wavelength 350nm, the width of the slit 5nm, the plasmid concentration of 0.07-1.75 mu g/ml in the range of good linearity, the recovery rate is 97.6-99.9%, the precision is high, and the chitosan and its derivative quaternized chitosan as the carrier material for the preparation of empty white rice grains, the key factor for the formation of nanoparticles: shell polymerization The concentration of sugar solution, pH value, N/P, TPP concentration, the particle size and zeta potential of nanoparticles were investigated. Based on the above conclusion, the formulation conditions of chitosan nanoparticles loaded with DNA were optimized. The formulation conditions were as follows: the concentration of CS solution was 0.25mg/ml, the buffer system pH was 5.5, TPP concentration solution 0.02mM, pVAX1-wapA150ng/ micron dissolved. The liquid concentration was 5mg/ml, the buffer system pH was 5, the TPP concentration solution 0.02mM, pVAX1-wapA300ng/ micron L. prepared the homogeneous particle size of the DNA vaccine. The nanoparticle size of.CSTM nanoparticles was 222.5nm, Zeta potential was 19.6mV, the encapsulation efficiency was 87.66%. For 91.24%, the drug loading was 34.22%.
On this basis, the fourth part of the experiment was carried out in the fourth part of the experiment. The transfection of the loaded plasmid nanoparticles was used to detect the transcriptional expression level of the plasmid encoding wapA gene by Realtime-PCR. The results showed that the naked plasmid pVAX1-wapA directly transfected cells almost did not detect the mRNA transcription of the wapA gene. The transfected pVAX1-wapA was co transfected with lipo2000. The level of transcription was 1, and the mRNA level of wapA gene was 0.87 after the pVAX1-wapA transfected by quaternated chitosan nanoparticles, which was 3.18 times the pVAX1-wapA transcription level of chitosan nanoparticles. The results of cell transfection in vitro showed that the chitosan nanoparticles and quaternated chitosan nanoparticles both promoted pVAX1-wapA into the cells. Work is transcribed and naked plasmids are almost unable to enter cells for normal transcription and translation.
In the fifth part of this paper, the fifth part of the experiment first used the pre constructed DNA vaccine and the drug delivery system to immunize normal mice. The saliva IgA and serum IgG level of the mice were detected by ELISA. The results showed that the DNA vaccine could successfully stimulate the immune system in the mice by means of the intramuscular injection, and the level of IgG antibody was raised, which proved that the recombinant plasmid pVAX1-wapA was constructed. The immunogenicity of the chitosan nanoparticles delivery system could improve the immunogenicity of the vaccine, but the intramuscular injection of the immunization pathway did not induce mucosal immunity. The experimental results did not observe the significant increase in the level of IgA antibody. The ELISA method was established to determine the specific IgG and IgA antibodies against wapA protein. In order to further confirm the anti caries effect of the DNA vaccine we constructed and whether it could effectively stimulate the mucosal immunity, the caries model of the fixed bacteria rats was established by cariogenic bacteria colonization and cariogenic feed, and the nasal cavity immunization was increased. The preventive and therapeutic effects of the new anti caries vaccine on the dental caries were evaluated. The results of animal model caries showed that the degree of dental caries was the lowest in the nasal cavity immune DNA vaccine nanoparticles group, and the level of saliva IgA and serum IgG antibody in the group also increased significantly. The caries loss of the DNA and DNA loaded DNA vaccine group was also lower, but the score was higher than that of the nasal cavity immunization group carrying the DNA vaccine and the DNA epidemic in the group. Although the vaccine and loaded DNA vaccine caused a high level of serum IgG, the level of saliva IgA was lower than that of the nasal immune group. The results show that the quaternized chitosan nanoparticles have good nasal immune effect and are suitable as the carrier of nasal immunity.
To sum up, a new recombinant plasmid pVAX1-wapA is constructed to prevent caries from a new anti caries antigen wapA, and it is revealed that wapA protein can be used as a anti caries target. Chitosan nanoparticles are used to improve the immune efficiency of DNA vaccine by using chitosan nanoparticles, which are non-toxic, safe and effective. The use of chitosan can promote the adhesion of DNA mucous membrane. The effect of immunization on the dental caries vaccine to improve the mucosal immune effect of DNA vaccine against dental caries vaccine is better than that of the intramuscular injection. The CSTM nanoparticles loaded with pVAX1-wapA have high efficiency and safety, which shows good application potential.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

【参考文献】