DNA防龋疫苗联合蛋白质疫苗诱导小鼠免疫应答的研究

目的:研究以一种新型免疫策略即追加相应蛋白质抗原来加强免疫DNA疫苗的免疫效果。方法:重组质粒pCIA-P经鼻腔滴注途径免疫小鼠,并以其相应抗原rPAc蛋白或rPAc蛋白及黏膜佐剂rCTB经鼻腔滴注加强免疫,ELISA法检测血清和唾液中特异性抗体水平。结果:以rPAc蛋白或rPAc蛋白及黏膜佐剂rCTB加强免疫可显著提高唾液中IgA型特异性抗体和血清IgG型特异性抗体水平。并且以rPAc蛋白和黏膜佐剂rCTB加强免疫组产生了最高水平的唾液IgA型特异性抗体和血清IgG型特异性抗体。结论:DNA防龋疫苗联合蛋白质疫苗可有效增强DNA防龋疫苗免疫效果。     

重组亚单位和合成肽防龋疫苗的免疫效能

目的：比较重组亚单位rPAc防龋疫苗和合成肽疫苗诱导小鼠特异性体液免疫和黏膜免疫水平。方法：以rPAc疫苗和合成肽疫苗分别滴鼻免疫小鼠,2、4周后加强免疫,分别收集0、2、4、6、8、10、12周的血清和唾液,用酶联免疫吸附实验（ELISA）检测血清和唾液中特异性抗体的水平。结果：以rPAc防龋疫苗免疫小鼠可产生较合成肽疫苗免疫小鼠更显著的黏膜免疫反应,两组比较差异有显著性（P〈0．05）。结论：重组亚单位rPAc防龋疫苗相对于合成肽疫苗具有更明显的黏膜免疫效果。     

防龋基因疫苗pcDNA3-PAc不同途径免疫BALB/c小鼠的实验研究

目的:观察防龋基因疫苗pcDNA3- PAc经不同途径免疫BALB/c小鼠后的免疫效果.方法: 重组质粒pcDNA3- PAc经颌下腺区皮下注射、股四头肌注射和鼻腔黏膜滴注免疫BALB/c小鼠,共免疫2 次,每次间隔2 周.分别于首次免疫前1 d和免疫后第1、2、4 周采集血液和唾液样品,采用酶联免疫吸附方法检测血清特异性IgG抗体和唾液特异性IgA抗体.结果: 各实验组免疫后1 周血清和唾液中特异性抗体水平开始升高,第4 周时达最高水平.颌下腺区皮下注射免疫产生的唾液IgA和股四头肌注射免疫诱导的血清IgG抗体水平明显高于其它实验组(P<0.05).颌下腺区皮下注射免疫和鼻腔黏膜滴注免疫可诱导较高水平的唾液IgA和一定水平的血清IgG;股四头肌注射免疫诱导的血清特异性IgG抗体水平较高(P<0.05),而唾液IgA抗体水平不如颌下腺区皮下注射免疫和鼻腔黏膜滴注免疫(P<0.05).结论: 防龋基因疫苗pcDNA3- PAc能够诱导动物机体产生有效的系统免疫应答和黏膜免疫应答.     

AddaVax经不同免疫途径对防龋疫苗的佐剂效应

目的：研究3种不同免疫途径下,防龋亚单位疫苗联合AddaVax佐剂接种小鼠所产生的免疫效果。方法：以rPAc与AddaVax混合物经鼻腔滴注、颊黏膜下注射及颈背部皮下注射等3种途径免疫小鼠,并以rPAc行鼻腔滴注免疫,ELISA法检测血清和唾液中的特异性抗体水平。结果：rPAc＋AddaVax皮下及颊黏膜下注射方式均可显著提高小鼠血清中抗PAcIgG水平;rPAc＋AddaVax颊黏膜下注射方式可有效提高小鼠唾液中抗PAcIgA水平。结论：rPAc与AddaVax联合经皮下及颊黏膜下注射方式可有效提高防龋疫苗的免疫效果。     

中试防龋DNA疫苗pGJA-P/VAX免疫小鼠的实验研究

目的：评价中试防龋DNA疫苗pGJA-P/VAX诱导小鼠体内特异性免疫应答的效果。方法：分别由武汉生物制品研究所中试制备和实验室采用德国Qiagen公司去内毒素质粒大量提取试剂盒手工提取防龋DNA疫苗pGJA-P/VAX;两种方法制备的质粒分别于0周,2周经肌肉、鼻腔免疫小鼠,并以鞭毛蛋白作为黏膜佐剂与质粒同时经鼻腔免疫小鼠,PBS及空载体pVAX1作为对照。免疫前开始每两周收集小鼠血清和唾液样本,ELISA定量检测血清及唾液中特异性抗体含量。结果：中试质粒和手提质粒肌注时均能够诱导血清特异性IgG,但诱导唾液特异性sIgA的能力较为有限。在与黏膜佐剂鞭毛蛋白同时滴鼻时,中试质粒可诱导较为明显的血清特异性IgG和唾液特异性sIgA。在相同的免疫条件下,中试质粒诱导的特异性抗体水平低于手提质粒组,但二者无显著性差异。结论：中试防龋DNA疫苗pGJA-P/VAX能够诱导小鼠体内特异性体液免疫应答。     

DNA防龋疫苗不同途径免疫BALB/c小鼠的实验研究

目的：观察编码pac结构基因A—P片段的重组质粒pCIA—P以不同途径免疫BALB／c小鼠后的特异性免疫反应。方法：重组质粒pCIA—P经鼻腔滴注和颌下腺区皮下腺注射两种途径免疫小鼠，ELISA法检测血清和唾液中特异性抗体水平动态变化。结果：滴鼻法和皮下注射法免疫后唾液IgA型特异性抗体和血清IgG型特异性抗体均明显升高，并持续数周。且滴鼻法比皮下注射法诱导唾液IgA型特异性抗体要早。结论：重组质粒pCIA—P是一种有效的免疫原，滴鼻法和颌下区皮下注射法接种防龋DNA疫苗都可有效诱导机体全身及局部黏膜免疫应答。而滴鼻法较皮下注射法能更早诱导局部黏膜免疫应答。     

基因重组乳链球菌防龋疫苗免疫定菌鼠的实验研究

目的为了阐明基因重组乳链球菌防龋疫苗的免疫效能。方法采用携带有变形链球菌表面蛋白（PAc）结构基因pac的重组乳链球菌防龋疫苗HL107通过灌胃、皮下注射、粘膜下注射三种途径对50只定菌Sprague－Dawley大鼠进行免疫，并以酶联免疫吸附试验进行鼠血清和唾液中抗PAc－IgG、IgA测定。结果通过灌胃进行免疫接种HL107的大白鼠血清中抗PAc－IgG、IgA抗体滴度和唾液中抗PAc－IgA抗体滴度明显高于乳链球菌LM0230免疫组和空白免疫组（P＜0.01）。结论基因重组乳链球菌具有变形链球菌表面蛋白的免疫原性，能够刺激定菌鼠产生特异性免疫反应和免疫表达产物。     

两种GTF氨基催化区改良防龋质粒免疫小鼠的实验研究

目的：通过比较编码表兄链球菌葡糖基转移酶氨基催化区CAT不同形式基因序列的两种改良防龋质粒免疫BALB/c小鼠后激发的特异性抗体水平差异,初步评价不同形式CAT在DNA防龋疫苗中的应用潜力。方法：利用分子克隆技术构建两种改良防龋质粒,在靶向防龋DNA疫苗pGJA-P/VAX中插入表兄链球菌OMZ176GTF-I CAT全基因序列构建pGJGAC/VAX;将基因公司合成的共计165bp的串联重复5次的GTF-I氨基催化区核心保守序列克隆到pGJA-P/VAX中构建pGJGA-5C/VAX。转染CHO细胞系,检测其表达。两种改良质粒经鼻腔滴注免疫BALB/c小鼠,检测血清和唾液中的特异性抗PAc,抗GLU和抗CAT抗体水平。结果：重组质粒的基因序列与预期相符。不同形式CAT基因序列的两种改良防龋质粒均可在真核细胞正确表达。pGJGAC/VAX和pGJGA-5C/VAX免疫动物后血清和唾液特异性抗PAc、抗GLU和抗CAT抗体均显著高于空载体pVAX1免疫组（P〈0.05）。第10、12和14周,pGJGAC/VAX免疫组小鼠的血清特异性抗CAT抗体显著高于pGJGA-5C/VAX组（P〈0.05）。两组间唾液特异性抗体未见显著性差异。pGJGA-5C/VAX在小鼠体内激发特异性抗体的速度较pGJGAC/VAX略快。结论：本实验构建的增加表兄链球菌GTF氨基催化区的两种改良防龋质粒,可在真核细胞中正确表达,两种质粒有效地诱导黏膜和系统体液免疫反应,pGJGAC/VAX和pGJGA-5C/VAX各有优势。     

重组变异链球菌表面蛋白的可溶性表达及抗体制备

目的探讨重组变异链球菌表面蛋白（rPAc）在大肠杆菌中可溶性表达的最佳诱导条件并制备其多克隆抗体。方法通过改变pET20b（+）-AP/BL21（DE3）plysS工程菌的培养条件,提高rPAc可溶性表达水平。用纯化的rPAc免疫BALB/c小鼠制备多克隆抗体,通过酶联免疫吸附测定（ELISA）和Western blot法鉴定抗体的免疫学活性。结果pET20b（+）-AP/BL21（DE3）plysS工程菌在Luria-Bertani（LB）培养基（pH值为7.2）上培养,至表示菌体密度的光密度值OD600 nm=0.6时加入1.0 mmol·L-1异丙基-β-D-硫代吡喃半乳糖苷（IPTG）,30 ℃诱导培养4 h,rPAc的可溶性表达量最高。以纯化的rPAc免疫BALB/c小鼠,制备抗rPAc多克隆抗体,ELISA法测定抗体效价为1∶6 000,Western blot法鉴定出该抗体可特异性识别rPAc。结论rPAc高效可溶性表达和抗rPAc多克隆抗体的制备为DNA初次免疫-蛋白质加强免疫策略的深入研究奠定了必要的物质基础。     

靶向融合防龋DNA疫苗pGJA-P免疫兔的实验研究

目的　体外检测靶向融合防龋DNA疫苗pGJA P的免疫反应性 ;与非靶向融合防龋DNA疫苗 pGLUA P进行比较 ,评价其增强疫苗免疫效能的能力。 方法　将 pGJA P转染CHO细胞 ,蛋白质免疫印迹实验检测重组蛋白抗体免疫反应性。分 5组免疫兔 :pGJA P经肌肉注射组 (A组 ) ;pGJA P经鼻黏膜免疫组 (B组 ) ;pGLUA P经肌肉注射 (C组 ) ;pGLUA P经鼻黏膜免疫组 (D组 )和pCI载体经股四头肌注射组 (E组 )。酶联免疫吸附实验检测血清及唾液中的特异性抗体。用收集的血清进行葡糖基转移酶 (GTF)合成水不溶性葡聚糖抑制实验。结果　pGJA P表达的重组蛋白可以与抗GTF抗体反应。A组血清特异性IgG抗体水平远高于C组 (P <0 0 1) ;B组唾液特异性IgA抗体水平显著高于D组 (P <0 0 1) ;A组同样诱导了高水平的唾液特异性IgA抗体。A组的免疫血清抑制GTF活性的能力最强。结论　pGJA P具备GTF的免疫反应性 ;并较pGLUA P有更强的诱导系统和黏膜免疫反应及抑制GTF的能力。     

Flagellin enhances saliva IgA response and protection of anti-caries DNA vaccine

We and others have shown that anti-caries DNA vaccines, including pGJA-P/VAX, are promising for preventing dental caries. However, challenges remain because of the low immunogenicity of DNA vaccines. In this study, we used recombinant flagellin protein derived from Salmonella (FliC) as a mucosal adjuvant for anti-caries DNA vaccine (pGJA-P/VAX) and analyzed the effects of FliC protein on the serum PAc-specific IgG and saliva PAc-specific IgA antibody responses, the colonization of Streptococcus mutans (S. mutans) on rat teeth, and the formation of caries lesions. Our results showed that FliC promoted the production of PAc-specific IgG in serum and secretory IgA (S-IgA) in saliva of rats by intranasal immunization with pGJA-P/VAX plus FliC. Furthermore, we found that enhanced PAc-specific IgA responses in saliva were associated with the inhibition of S. mutans colonization of tooth surfaces and endowed better protection with significant fewer caries lesions. In conclusion, our study demonstrates that recombinant FliC could enhance specific IgA responses in saliva and protective ability of pGJA-P/VAX, providing an effective mucosal adjuvant candidate for intranasal immunization of an anti-caries DNA vaccine.     

靶向牙周炎DNA疫苗免疫原性及免疫效能的实验研究

目的：检测靶向牙周炎DNA疫苗pCTLA4-FimA的免疫反应性,并评价其免疫保护能力。方法：分3组免疫BALB/c小鼠：pCTLA4-FimA鼻黏膜免疫组,非靶向牙周炎DNA疫苗pFimA鼻黏膜免疫组,pCI载体鼻黏膜免疫组。酶联免疫吸附实验检测血清及唾液中特异性抗体水平。建立小鼠牙周炎模型,检测牙槽骨水平吸收程度。结果：与pFimA免疫组相比,pCTLA4-FimA免疫组诱导了显著增强的血清特异性IgG抗体和唾液特异性IgA抗体水平（P〈0.01）。与免疫前相比,3实验组牙槽骨均有吸收,其中pCTLA4-FimA免疫组牙槽骨水平吸收程度最低。结论：靶向牙周炎DNA疫苗pCTLA4-FimA能有效诱导机体的免疫反应,并抑制牙周炎的发展,效果优于非靶向牙周炎DNA疫苗pFimA。     

Studies on the induction of the humoral immune responses to Bacteroides gingivalis fimbrial antigen in mice

Serum and salivary antibody responses to Bacteroides gingivalis fimbriae administered either orally or subcutaneously (s.c.) with or without an adjuvant in various strains of mice were examined in this study. Following results were obtained. 1) Oral administration of B. gingivalis fimbriae with GM-53 as an adjuvant in liposomes, but not in Tris-HCl buffer, definitely enhanced the fimbriae-specific IgG responses, mainly IgG1 followed by IgG2b, IgG2a and IgG3 in serum and IgA response in saliva of BALB/c mice. On the other hand, s.c. injection of fimbriae with GM-53 or MDP-Lys (L18) also raised the fimbriae-specific IgG followed by IgA and IgM responses in serum, and both IgA and IgG responses in saliva of BALB/c mice. Oral immunization was less effective than s.c. injection in terms of the production of serum antibody in the mice. However, the level of salivary antibody of mice injected s.c. was similar to that of mice immunized orally. 2) High anti-fimbriae antibodies in serum were maintained in BALB/c mice immunized orally with fimbriae and GM-53 in liposomes for approximately 7 months after the primary immunizations. Oral administration also induced and held the fimbriae-specific IgA response in saliva for at least 6 months after the primary immunizations. The levels of fimbriae-specific IgA in saliva after the second boosters on days 123 and 124 were higher than those after the primary ones on days 27 and 28. 3) Among various strains of mice immunized orally with fimbriae and GM-53 in liposomes, BALB/c and DBA/2 mice (H-2d) significantly produced high levels of both serum IgG and salivary IgA antibodies specific for fimbriae. Furthermore, B10.D2 mice (H-2d) were responders followed by B10.BR (H-2k), while C57BL/10 mice (B10, H-2b) were low responders to the fimbriae. These results show that the combined use of fimbriae together with an adjuvant results in a sharply increased IgA antibody response in saliva and a predominantly stimulated IgG antibody in serum, and it was suggested that these responses are restricted by H-2 haplotype.     

基因重组乳链球菌防龋疫苗口服免疫孕兔的实验研究

作者研究了重组链球菌ＨＬ１０７口服免疫孕兔的效果。使用酶联免疫吸附法测定口服免疫前后血清、唾液和乳汁中抗表面蛋白抗原ＩｇＧ、ＩｇＡ抗体水平。结果表明口服免疫孕兔后血清和乳汁中特异性抗表面蛋白抗原的ＩｇＧ水平明显升高，唾液和乳汁中抗表面蛋白抗原的ＩｇＡ水平明显升高，提示重组乳链球菌ＨＬ１０７具有变形链球菌表面蛋白抗原的免疫原性，能够激发针对表面蛋白质抗原的系统免疫和局部粘膜的免疫反应。     

Salivary gland delivery of pDNA-cationic lipoplexes elicits systemic immune responses

OBJECTIVE: To test the ability of two cationic lipoplexes, Vaxfectin and GAP-DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands. METHODS: Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP-DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti-influenza NP-specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme- linked immunosorbent assay (ELISA). Cytotoxic T-cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes. RESULTS: Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP-DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P < 0.05). No rats developed IgA antibodies to NP protein in saliva. Animals receiving plasmid encoding hGH and either lipoplex expressed significantly higher amounts of hGH compared with those receiving the hGH plasmid and water. Although hGH expression was higher in the animals receiving pDNA/Vaxfectin (approximately 30-fold > pDNA/water), the difference with those receiving pDNA/GAP-DLRIE/DOPE (approximately 10-fold > pDNA/water) was not significant. CONCLUSIONS: Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization.