本文選題：齲齒 + DNA疫苗　； 參考：《第二軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：近年來(lái)，隨著人們生活質(zhì)量的提高和老齡化程度的加深，越來(lái)越多的人開始關(guān)注口腔衛(wèi)生。齲齒的發(fā)病率僅次于流感。輕度的齲齒影響咀嚼功能，重度齲齒可以引起根尖周病、牙髓病、頜骨炎癥等并發(fā)癥，嚴(yán)重影響人類全身健康和生存質(zhì)量。由于我國(guó)人口眾多、醫(yī)療資源有限，研制易于普及的齲病疫苗以防治齲病、降低齲病發(fā)病率對(duì)于保障國(guó)民健康顯得尤為重要。 變形鏈球菌（S. mutants）是公認(rèn)的主要致齲因素之一，wapA蛋白是變形鏈球菌的表面蛋白，參與變形鏈球菌在宿主牙齒上的聚集粘附和菌斑形成。 本文第一部分把變形鏈球菌UA159的編碼wapA蛋白的全長(zhǎng)基因序列重組到真核表達(dá)載體pVAX1上構(gòu)建抗齲DNA疫苗pVAX1-wapA，建立其原核大腸桿菌（E.coli）表達(dá)系統(tǒng)。通過(guò)酶切與測(cè)序比對(duì)鑒定，構(gòu)建的質(zhì)粒序列正確，無(wú)移碼，突變，堿基缺失等現(xiàn)象。并提取純化了大量的pVAX1-wapA以用于后續(xù)的處方設(shè)計(jì)和體內(nèi)外評(píng)價(jià)實(shí)驗(yàn)。 本文第二部分構(gòu)建了wapA蛋白的原核大腸桿菌表達(dá)系統(tǒng)，對(duì)時(shí)間，溫度以及IPTG濃度等表達(dá)條件進(jìn)行優(yōu)化，結(jié)果顯示最佳誘導(dǎo)表達(dá)條件是0.1mM IPTG，37℃，5h。通過(guò)親和凝膠過(guò)濾的層析方法純化得到了5.2mg，純度為90%的wapA重組蛋白。 本文第三部分實(shí)驗(yàn)以甲基丙烯酰氧乙基三甲基氯化銨（TMAEMC）修飾殼聚糖（CS），通過(guò)自由基聚合反應(yīng)合成了水溶性良好的季銨化殼聚糖(CSTM)。通過(guò)1H-NMR和FT-IR對(duì)其結(jié)構(gòu)進(jìn)行了確證。合成的季銨化殼聚糖季銨化程度為52%。建立了質(zhì)粒定量的熒光分光光度法，最佳發(fā)射波長(zhǎng)464nm，最佳激發(fā)波長(zhǎng)350nm，狹縫寬度5nm，質(zhì)粒濃度0.07-1.75μg/ml范圍內(nèi)線性良好，回收率在97.6-99.9%，精密度高。并以殼聚糖及其衍生物季銨化殼聚糖為載體材料制備空白納米粒，對(duì)納米粒形成的關(guān)鍵因素：殼聚糖溶液濃度，pH值，N/P，TPP濃度，以納米粒的粒徑和zeta電位為指標(biāo)進(jìn)行考察�；谏鲜鼋Y(jié)論，對(duì)包載DNA的殼聚糖納米粒的處方條件進(jìn)行優(yōu)化，最終確定處方條件為：CS溶液濃度為0.25mg/ml，緩沖體系pH為5.5，TPP濃度溶液0.02mM，pVAX1-wapA150ng/μl。CSTM溶液濃度為5mg/ml，緩沖體系pH為5.0，TPP濃度溶液0.02mM，pVAX1-wapA300ng/μl。制備了包載DNA疫苗粒徑均一，形態(tài)圓整的納米粒。CSTM納米粒粒徑為222.5nm，Zeta電位為19.6mV，包封率為87.66%，載藥量為4.96%。制備的CS納米粒粒徑為219.2nm，Zeta電位為24.7mV，，包封率為91.24%，載藥量為34.22%。 在此基礎(chǔ)上，本文第四部分實(shí)驗(yàn)使用包載質(zhì)粒的納米粒進(jìn)行細(xì)胞轉(zhuǎn)染，通過(guò)Realtime-PCR檢測(cè)細(xì)胞對(duì)質(zhì)粒編碼wapA基因的轉(zhuǎn)錄表達(dá)水平。結(jié)果顯示裸質(zhì)粒pVAX1-wapA直接轉(zhuǎn)染的細(xì)胞幾乎檢測(cè)不到wapA基因的mRNA轉(zhuǎn)錄。以與lipo2000共轉(zhuǎn)染的pVAX1-wapA的轉(zhuǎn)錄水平為1，季銨化殼聚糖納米粒包載的pVAX1-wapA轉(zhuǎn)染細(xì)胞后wapA基因的mRNA水平為0.87，是殼聚糖的納米粒包載的pVAX1-wapA轉(zhuǎn)錄水平的3.18倍。體外細(xì)胞轉(zhuǎn)染結(jié)果表明，殼聚糖納米粒和季銨化殼聚糖納米粒均可以促進(jìn)pVAX1-wapA進(jìn)入細(xì)胞，并成功被轉(zhuǎn)錄。而裸質(zhì)粒幾乎無(wú)法進(jìn)入細(xì)胞進(jìn)行正常的轉(zhuǎn)錄翻譯。 本文第五部分實(shí)驗(yàn)首先使用前期構(gòu)建的DNA疫苗和給藥系統(tǒng)免疫正常小鼠，采用ELISA檢測(cè)小鼠的唾液IgA和血清IgG水平。結(jié)果發(fā)現(xiàn)，DNA疫苗通過(guò)肌注途徑可以成功激發(fā)小鼠免疫系統(tǒng)，產(chǎn)生IgG抗體水平的升高，證明了構(gòu)建的重組質(zhì)粒pVAX1-wapA具有免疫原性。并且殼聚糖納米粒遞送系統(tǒng)可以提高疫苗肌注的免疫原性，但肌注免疫途徑并不能誘導(dǎo)粘膜免疫，實(shí)驗(yàn)結(jié)果沒有觀察到IgA抗體水平的顯著升高。建立了ELISA法用于測(cè)定抗wapA蛋白特異性IgG和IgA抗體，方法批內(nèi)和批間的變異系數(shù)均小于10%，重現(xiàn)性好。為了進(jìn)一步證實(shí)我們構(gòu)建的DNA疫苗的抗齲效果和能否有效激發(fā)粘膜免疫，采用致齲菌定植和致齲飼料建立定菌大鼠齲齒模型，增加了鼻腔免疫途徑，對(duì)前期構(gòu)建的新型抗齲齒疫苗對(duì)齲齒的預(yù)防和治療作用進(jìn)行評(píng)價(jià)。動(dòng)物模型齲損評(píng)分結(jié)果顯示，鼻腔免疫DNA疫苗納米粒組齲損程度最低，該組的唾液IgA和血清IgG抗體水平也顯著提高。肌注DNA疫苗和包載DNA疫苗的納米粒組齲損也有降低，但評(píng)分都要高于包載DNA疫苗的納米粒的鼻腔免疫組，肌注DNA疫苗和包載DNA疫苗雖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組。本部分結(jié)果說(shuō)明季銨化殼聚糖納米粒具有良好的鼻腔免疫效果，適合作為鼻腔免疫的載體。 綜上所述，本文針對(duì)一種新的抗齲抗原wapA，構(gòu)建了一種全新的重組質(zhì)粒pVAX1-wapA用于防齲，并揭示wapA蛋白可以作為一個(gè)抗齲靶點(diǎn)。利用殼聚糖納米粒無(wú)毒、安全、有效的穿細(xì)胞能力，提高了DNA疫苗的免疫效率。利用殼聚糖可促進(jìn)DNA粘膜粘附和吸收，同時(shí)具有免疫佐劑的作用。針對(duì)齲齒疫苗采用鼻腔接種，提高DNA疫苗黏膜免疫效果，相比于肌注免疫顯示更強(qiáng)的抗齲活性。包載pVAX1-wapA的CSTM納米粒作為鼻腔免疫疫苗具有高效、安全的特點(diǎn)，顯示出良好的應(yīng)用潛力。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

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