本文選題：肺炎鏈球菌 + 疫苗��； 參考：《重慶醫(yī)科大學》2011年碩士論文

【摘要】：目的 肺炎鏈球菌(streptococcus pneumoniae, S.pn)是引起嚴重侵襲性感染和上呼吸道感染最重要的病原體之一, WHO估計全球每年約有160萬人死于肺炎鏈球菌引起的各種疾病,約100萬病例為5歲以下兒童,而90%以上的死亡病例在發(fā)展中國家;隨著耐藥菌株的不斷出現,肺炎鏈球菌感染的治療面臨越來越多的問題,有效的疫苗成為預防其感染的一個重要手段。 目前,免疫途徑的選擇正在成為疫苗開發(fā)需要考慮的問題,與傳統(tǒng)的肌肉注射免疫方式相比,粘膜免疫在產生局部免疫反應的同時,還可以刺激系統(tǒng)性免疫反應,對許多感染性疾病的防御都起重要作用,因此,WHO將粘膜免疫定為兒科疫苗發(fā)展的方向。 溶血素(Pneumolysin, Ply)為肺炎鏈球菌的重要毒素之一,幾乎存在于所有血清型的S.pn中,具有很好的保守性,最新研究報道顯示,Ply在菌體表面存在表達。但是,由于Ply具有細胞毒性,必須對其優(yōu)化。本課題組前期研究表明,第146位丙氨基酸缺失后可以導致Ply毒力和溶血活性完全喪失,并能誘導宿主產生保護性抗體,因此,突變的Ply是肺炎鏈球菌良好的疫苗候選蛋白,本課題擬研究ΔA146 Ply粘膜免疫對肺炎鏈球菌感染的保護作用。 方法 利用重組蛋白原核表達技術對第146位氨基酸缺失的Ply蛋白(ΔA146 Ply)進行體外表達,重組蛋白經粘膜途徑免疫BALB/C小鼠,制備ΔA146 Ply特異性抗血清。SDS-PAGE及Western blot分析目的蛋白的表達及在六株常見致病性肺炎鏈球菌中的保守性,間接ELISA檢測血清及唾液中ΔA146 Ply特異性抗體效價,并分析其產生IgG的亞型,同時,無菌操作獲得免疫后小鼠脾細胞,檢測其培養(yǎng)上清中相應的細胞因子水平。19F型S.pn鼻腔感染免疫后小鼠,取其鼻腔灌洗液和肺勻漿,鋪板計數其中定植的細菌數量;致病性肺炎鏈球菌NCTC7466,CMCC(B)31436、CMCC(B)31207及CMCC(B)31614菌株經鼻腔感染免疫后小鼠,檢測重組Ply蛋白對其在小鼠鼻咽部及肺部感染的保護作用并評價其主動免疫保護效果。進行體外抗黏附實驗,檢測ΔA146 Ply蛋白及其抗血清是否對肺炎鏈球菌R6黏附A549細胞具有抑制作用。 結果 包含肺炎鏈球菌ply(146位氨基酸突變)序列的重組工程菌BL21(DE3)經IPTG誘導得到大量以可溶形式表達的重組蛋白,經Ni-NTA親和層析純化,SDS-PAGE檢測發(fā)現得到了純度95%的純化蛋白。Western blot檢測發(fā)現,Ply在六株常見的致病性肺炎鏈球菌NCTC7466,TIGR4,CMCC(B)31436、CMCC(B)31207,CMCC(B)31614及CMCC(B)31693中具有很好的保守性。重組蛋白經粘膜免疫BALB/C小鼠,檢測其血清及唾液中特異性抗體發(fā)現,血清中ΔA146Ply特異性IgG效價高達5.12×10~5,唾液中IgG及IgA效價分別達到4.0×10~3和4.8×10~3;利用Santa公司羊抗鼠IgG1,IgG2a,IgG2b,IgG3二抗亞型檢測免疫后血清中IgG亞型,結果顯示,重組蛋白ΔA146 Ply粘膜免疫BALB/C小鼠主要刺激宿主產生IgG1,和IgG2b為主,提示宿主產生的免疫反應主要為Th2型。檢測免疫后小鼠脾細胞培養(yǎng)上清中相應細胞因子水平發(fā)現,ΔA146 Ply粘膜免疫后刺激宿主產生了高水平的IL-17A,劑量高達678.55±189(pg/ml),峰值出現于刺激后48小時。CMCC(B)31693抗定植實驗顯示,ΔA146 Ply粘膜免疫后肺炎鏈球菌在小鼠鼻咽部及肺部的定植降低10-20倍(p0.05)。致病性肺炎鏈球菌NCTC7466 , CMCC(B)31436、CMCC(B)31207及CMCC(B)31614菌株經鼻腔感染免疫后小鼠,檢測其在小鼠鼻咽部及肺部的細菌數量發(fā)現,肺炎鏈球菌CMCC(B)31436及CMCC(B)31614在小鼠鼻咽部的定植降低20倍左右(p0.05),肺炎鏈球菌NCTC7466,CMCC(B)314367及CMCC(B)31614在小鼠肺內的定植降低20-30倍(p0.05)。主動免疫保護實驗顯示,重組蛋白ΔA146 Ply粘膜免疫可以有效延長小鼠生存時間,提高其生存率,其對肺炎鏈球菌NCTC7466,CMCC(B)31436、CMCC(B)31614及CMCC(B)31207的保護率分別為50%,41.7%,50%,41.7%。體外抗黏附實驗結果顯示,ΔA146 Ply特異性抗血清100倍、500倍、2500倍和10000倍稀釋時,對肺炎鏈球菌R6對黏附A549細胞的抑制率分別為65%、60%、55%和35%;重組ΔA146 Ply蛋白在1μg/ml, 10μg/ml, 20μg/ml, 50μg/ml時對肺炎鏈球菌R6黏附A549細胞的抑制率分別為15%、20%、25%和 30%。 結論 上述結果提示,ΔA146 Ply蛋白經粘膜免疫BALB/C小鼠可有效降低肺炎鏈球菌在宿主鼻咽部和肺部的定植,其保護作用可能依賴于高水平的IgA抗體及IL-17A;同時ΔA146 Ply蛋白粘膜免疫能有效保護宿主抵御四種致病性血清型S.pn的致死性感染,其保護作用可能依賴于小鼠體液免疫激活而產生的特異性抗體。
[Abstract]:objective
Streptococcus pneumoniae (S.pn) is one of the most important pathogens causing severe invasive infection and upper respiratory tract infection. WHO estimates that about 1 million 600 thousand people worldwide die from various diseases caused by Streptococcus pneumoniae every year. About 1 million cases are children under 5 years of age, and more than 90% of the deaths are in developing countries. With the continuous emergence of strains, the treatment of Streptococcus pneumoniae is facing more and more problems. Effective vaccines have become an important means to prevent infection.
At present, the selection of immune pathways is becoming a problem to be considered in the development of vaccine. Compared with the traditional intramuscular injection immunization, mucosal immunity can stimulate systemic immune response and play a important role in the defense of many infectious diseases. Therefore, WHO makes mucosal immunization a paediatric vaccine. The direction of development.
Pneumolysin (Ply) is one of the most important toxin of Streptococcus pneumoniae. It almost exists in all serotypes of S.pn and has good conservatism. The latest research report shows that Ply is expressed on the surface of the fungus. But, because Ply has cytotoxicity, it must be optimized. The previous study in this group showed that 146th bits of amino acid were deficient. After loss, the Ply toxicity and hemolytic activity can be completely lost and the host can induce protective antibodies. Therefore, the mutant Ply is a good candidate protein for Streptococcus pneumoniae. This topic is to study the protective effect of delta A146 Ply mucosal immunization on Streptococcus pneumoniae infection.
Method
The recombinant protein prokaryotic expression technique was used to express the 146th amino acid missing Ply protein (delta A146 Ply) in vitro. The recombinant protein was immunized with the mucosal pathway to immunization of BALB/C mice. The expression of delta A146 Ply specific antiserum.SDS-PAGE and Western blot analysis target protein and the conservatism in Six Common Pathogenic Streptococcus pneumoniae were prepared. The titer of the specific antibody of delta A146 Ply in serum and saliva was detected by ELISA, and the subtype of IgG was analyzed. At the same time, the spleen cells of mice after immunization were obtained by aseptic operation, and the corresponding cytokine level of.19F S.pn nasal infection in the culture supernatant was detected, and the nasal cavity lavage fluid and lung homogenate were obtained. The number of bacteria, Pathogenic Streptococcus pneumoniae NCTC7466, CMCC (B) 31436, CMCC (B) 31207 and CMCC (B) 31614 were immunized by nasal infection in mice. The protective effect of recombinant Ply protein on its nasopharynx and lung infection in mice was detected and its active immunity protection was evaluated. The anti adhesion test in vitro was conducted to detect the delta A146 Ply protein and its antiserum. Whether it has inhibitory effect on A549 cells adhered to Streptococcus pneumoniae R6.
Result
Recombinant recombinant strain BL21 (DE3) containing Streptococcus pneumoniae ply (146 amino acid mutation) sequence was induced by IPTG to obtain a large number of recombinant proteins expressed in soluble form. Purified by Ni-NTA affinity chromatography, SDS-PAGE detection found that the purity of purified protein.Western blot was detected by SDS-PAGE, and Ply was found in six common Pathogenic Streptococcus pneumoniae NCTC7. 466, TIGR4, CMCC (B) 31436, CMCC (B) 31207, CMCC (B) 31614 and CMCC (B) 31693 have good conservatism. The recombinant protein was immunized with the mucous membrane of the mucous membrane to detect the specific antibodies in the serum and saliva. The delta A146Ply specificity IgG titer of the serum was up to 5.12, and the potency of the saliva was 4 * and 4.8, respectively. NTA Sheep anti rat IgG1, IgG2a, IgG2b, IgG3 two anti subtypes were used to detect the IgG subtypes in the immune sera. The results showed that the recombinant protein Delta A146 Ply mucosal immune BALB/C mice mainly stimulated the host to produce IgG1, and IgG2b, suggesting that the host immune response was mainly Th2 type. The corresponding cell causes in the splenocytes culture supernatant of mice after immunization were detected. At the sub level, a high level of IL-17A was produced by the immune stimulation of the delta A146 Ply mucosa. The dose was up to 678.55 + 189 (pg/ml). The peak appeared at 48 hours after the stimulation, and.CMCC (B) 31693 anti colonization test showed that the colonization of Streptococcus pneumoniae in the nasopharynx and lungs of the mice decreased by 10-20 times (P0.05). The pathogenic pneumonic chain of Streptococcus pneumoniae was reduced by A146 Ply. The bacteria NCTC7466, CMCC (B) 31436, CMCC (B) 31207 and CMCC (B) 31614 were immunized by nasal infection, and the number of bacteria in the nasopharynx and lungs of mice was detected. The colonization of Streptococcus pneumoniae CMCC (B) 31436 and CMCC (B) 31614 in the nasopharynx of mice decreased by 20 times (P0.05), 314367 and 31614 were 31614 in the nasopharynx. The colonization of lung in mice decreased by 20-30 times (P0.05). The active immune protection experiment showed that the recombinant protein Delta A146 Ply mucosal immune can effectively prolong the survival time of mice and improve its survival rate. The protection rate of Streptococcus pneumoniae NCTC7466, CMCC (B) 31436, CMCC (B) 31614 and CMCC (B) 31207 is 50%, 41.7%, 50%, and 41.7%. in vitro adherence experimental knot, respectively. The results showed that the inhibition rates of Streptococcus pneumoniae R6 against adhesion A549 cells were 65%, 60%, 55% and 35% respectively when the delta A146 Ply specific antiserum was diluted 100 times, 500 times, 2500 times and 10000 times, and the inhibition rates of the recombinant Delta A146 Ply protein at 1 mu g/ml, 10 um g/ml, 20 g/ml and 50 mu g/ml respectively were 15%, 20%, and A549 cells respectively.
30%.
conclusion
These results suggest that A146 Ply protein can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lungs of the host by mucous membrane immunization, and its protective effect may depend on the high level of IgA antibody and IL-17A, while the delta A146 Ply protein mucosal immunization can effectively protect the host from the lethal infection of the four pathogenic serotype S.pn. The protective effect may depend on the specific antibody produced by humoral immune activation in mice.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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