致羔羊腹瀉奇異變形桿菌的分離鑒定及其ompA基因重組乳酸乳球菌口服疫苗研制
發(fā)布時(shí)間：2018-11-10 20:19
【摘要】：2015年春,山東滕州某規(guī)模化羊場(chǎng)新生羔羊出現(xiàn)嚴(yán)重的腹瀉,并且部分羔羊死亡。對(duì)該羊場(chǎng)病死羔羊進(jìn)行剖檢后,共分離到四株細(xì)菌(分別命名為PM1、PM2、PM3、PM4)。對(duì)所分離的細(xì)菌進(jìn)行形態(tài)特征、生化試驗(yàn)以及16S rDNA遺傳分析,判定該四株菌均為奇異變形桿菌。16S rDNA系統(tǒng)發(fā)育及同源性分析顯示:四株分離菌與其它11株(NCBI收錄)奇異變形桿菌的同源性為99%以上,其中與中國(guó)Hμ株、馬來西亞PPB3株以及印度BAB-199株位于一個(gè)分支上,且與中國(guó)的Hμ株的進(jìn)化關(guān)系最近;四株分離菌之間的同源性為100%,判斷為同一株菌。細(xì)菌生長(zhǎng)曲線、藥敏試驗(yàn)、毒力試驗(yàn)、毒素測(cè)定試驗(yàn)以及游散行為分析結(jié)果表明,該分離菌于14 h左右達(dá)到生長(zhǎng)穩(wěn)定期,且對(duì)環(huán)丙沙星、左氟沙星最為敏感;對(duì)小白鼠有強(qiáng)致病性,分離菌培養(yǎng)液的無菌濾液對(duì)小白鼠無毒性作用;在含0.5%瓊脂的LB平板上遷徙速度最快,且在含瓊脂1.5%和2.0%的LB平板上呈圓環(huán)樣周期運(yùn)動(dòng)。該研究結(jié)果為羊奇異變形桿菌病的防控提供了基礎(chǔ)。本試驗(yàn)構(gòu)建了含羊奇異變形桿菌外膜蛋白A(ompA)編碼基因的重組載體pNZ8149-ompA,并將其電轉(zhuǎn)入乳酸乳球菌NZ3900中。利用Westen blot檢測(cè)目的蛋白的表達(dá),間接免疫熒光試驗(yàn)及流式細(xì)胞儀檢測(cè)目的蛋白在乳酸乳球菌中的定位。將重組乳酸乳球菌灌胃BALB/c小鼠,分別于口服后的1、2、3、4、5、6、7 d之后取其十二指腸、空腸、回腸、盲腸的腸道沖洗液,通過平板菌落計(jì)數(shù)法檢測(cè)腸道內(nèi)的乳酸乳球菌定植情況。Western blot檢測(cè)到目的蛋白于49kDa處出現(xiàn)特異性條帶;間接免疫熒光試驗(yàn)檢測(cè)到重組乳酸乳球菌菌體表面出現(xiàn)綠色熒光;流式細(xì)胞儀檢測(cè)到重組乳酸乳球菌表面熒光強(qiáng)度明顯強(qiáng)于空質(zhì)粒對(duì)照組。重組乳酸乳球菌灌服小鼠后能在腸道黏膜的不同部位以一定比例存活,并于口服后6 d達(dá)到定植高峰,7 d后在十二指腸、空腸、回腸和盲腸定植率分別占第1 d的15.23%、24.19%、48.57%、40.07%。本試驗(yàn)表明羊奇異變形桿菌ompA能夠穩(wěn)定表達(dá)于乳酸乳球菌表面,且重組菌在小鼠腸道內(nèi)具有良好的定植能力,定植規(guī)律為回腸盲腸空腸十二指腸。這為研究乳酸乳球菌作為羊奇異變形桿菌口服疫苗及其對(duì)小鼠腸道免疫機(jī)理的影響提供試驗(yàn)基礎(chǔ)。以泰山松花粉多糖(TPPPS)作為口服疫苗佐劑,探究其對(duì)小鼠免疫功能作用的影響,結(jié)果表明,單純的重組乳酸乳球菌口服疫苗能誘導(dǎo)特異性IgA和IgG、IL-2、IL-4和IFN-γ的產(chǎn)生,促進(jìn)T淋巴細(xì)胞增殖,且免疫后小鼠可顯著抵抗奇異變形桿菌于腸道內(nèi)定植。此外,與單純重組乳酸乳球菌口服疫苗相比,TPPPS佐劑可誘導(dǎo)更高水平的IgA、IgG、IL-2、IL-4和IFN-γ,以及T淋巴細(xì)胞增殖反應(yīng)。綜上所述,乳酸乳球菌聯(lián)合TPPPS佐劑制備口服疫苗,提供了一種預(yù)防奇異變形桿菌感染的新型方法。
[Abstract]:In the spring of 2015, severe diarrhea and the death of some lambs occurred in a large scale sheep farm in Tengzhou, Shandong Province. Four strains of bacteria (named PM1,PM2,PM3,PM4) were isolated from the dead lamb. Morphological characteristics, biochemical tests and 16s rDNA genetic analysis were performed on the isolated bacteria. The phylogeny and homology analysis of 16s rDNA showed that the homology of the four isolates with other 11 strains (NCBI included) was more than 99%, and the homology was more than 99% with that of H 渭 strain in China. The Malaysian PPB3 strain and the Indian BAB-199 strain are located in one branch and have the closest evolutionary relationship with H 渭 strain in China. The homology of the four isolates was 100, which was determined to be the same strain. The results of bacterial growth curve, drug sensitivity test, virulence test, toxin test and swimming behavior analysis showed that the isolated bacteria reached the stable growth stage at about 14 h, and levofloxacin was the most sensitive to ciprofloxacin. It has strong pathogenicity to mice, and the aseptic filtrate of isolated bacteria culture medium has no toxic effect on mice. The migration rate was the fastest on the LB plate containing 0.5% Agar and the circular cycle motion was observed on the LB plate containing 1.5% Agar and 2.0% Agar. The results provide a basis for the prevention and control of Proteus mirabilis disease in sheep. In this study, the recombinant vector pNZ8149-ompA, containing A (ompA) encoding gene of Proteus mirabilis outer membrane protein was constructed and electrotransferred into NZ3900 of Lactococcus lactis. The expression of target protein was detected by Westen blot, the localization of target protein in Lactococcus lactis was detected by indirect immunofluorescence assay and flow cytometry. BALB/c mice were perfused with recombinant Lactococcus lactis. After oral administration of Lactococcus lactis, the intestinal irrigation fluid of duodenum, jejunum, ileum and cecum were collected after oral administration for 7 days. The colonization of Lactococcus lactis in intestinal tract was detected by plate colony counting method. The specific band of target protein was found in 49kDa by. Western blot. Indirect immunofluorescence assay detected green fluorescence on the surface of recombinant Lactococcus lactis, and flow cytometry showed that the fluorescence intensity of recombinant Lactococcus lactis was significantly stronger than that of blank plasmid control group. Recombinant Lactococcus lactis could survive in different parts of intestinal mucosa in certain proportion after oral administration, and reached the colonization peak at 6 days after oral administration, and 15.23 percent in duodenum, jejunum, ileum and cecum at 7 days after oral administration. 24.19 there are 48.57 and 40.07. The results showed that Proteus mirabilis ompA could be stably expressed on the surface of Lactococcus lactis, and the recombinant bacteria had good colonization ability in mouse intestine, and the colonization rule was jejunum duodenum of ileum caecum. This provides the experimental basis for the study of Lactococcus lactis as an oral vaccine of Proteus mirabilis and its effect on the intestinal immune mechanism of mice. The effects of Taishanpine Pollen Polysaccharide (TPPPS) as adjuvant on immune function in mice were investigated. The results showed that the recombinant Lactococcus lactis oral vaccine could induce specific IgA and IgG,IL-2,. The production of IL-4 and IFN- 緯 promoted the proliferation of T lymphocytes, and the immunized mice could significantly resist the colonization of Proteus mirabilis in the intestine. In addition, TPPPS adjuvant could induce higher levels of IgA,IgG,IL-2,IL-4 and IFN- 緯, as well as T lymphocyte proliferation, compared with recombinant lactococcus lactis oral vaccine. To sum up, Lactococcus lactis combined with TPPPS adjuvant to prepare oral vaccine provides a new method to prevent Proteus mirabilis infection.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.26
[Abstract]:In the spring of 2015, severe diarrhea and the death of some lambs occurred in a large scale sheep farm in Tengzhou, Shandong Province. Four strains of bacteria (named PM1,PM2,PM3,PM4) were isolated from the dead lamb. Morphological characteristics, biochemical tests and 16s rDNA genetic analysis were performed on the isolated bacteria. The phylogeny and homology analysis of 16s rDNA showed that the homology of the four isolates with other 11 strains (NCBI included) was more than 99%, and the homology was more than 99% with that of H 渭 strain in China. The Malaysian PPB3 strain and the Indian BAB-199 strain are located in one branch and have the closest evolutionary relationship with H 渭 strain in China. The homology of the four isolates was 100, which was determined to be the same strain. The results of bacterial growth curve, drug sensitivity test, virulence test, toxin test and swimming behavior analysis showed that the isolated bacteria reached the stable growth stage at about 14 h, and levofloxacin was the most sensitive to ciprofloxacin. It has strong pathogenicity to mice, and the aseptic filtrate of isolated bacteria culture medium has no toxic effect on mice. The migration rate was the fastest on the LB plate containing 0.5% Agar and the circular cycle motion was observed on the LB plate containing 1.5% Agar and 2.0% Agar. The results provide a basis for the prevention and control of Proteus mirabilis disease in sheep. In this study, the recombinant vector pNZ8149-ompA, containing A (ompA) encoding gene of Proteus mirabilis outer membrane protein was constructed and electrotransferred into NZ3900 of Lactococcus lactis. The expression of target protein was detected by Westen blot, the localization of target protein in Lactococcus lactis was detected by indirect immunofluorescence assay and flow cytometry. BALB/c mice were perfused with recombinant Lactococcus lactis. After oral administration of Lactococcus lactis, the intestinal irrigation fluid of duodenum, jejunum, ileum and cecum were collected after oral administration for 7 days. The colonization of Lactococcus lactis in intestinal tract was detected by plate colony counting method. The specific band of target protein was found in 49kDa by. Western blot. Indirect immunofluorescence assay detected green fluorescence on the surface of recombinant Lactococcus lactis, and flow cytometry showed that the fluorescence intensity of recombinant Lactococcus lactis was significantly stronger than that of blank plasmid control group. Recombinant Lactococcus lactis could survive in different parts of intestinal mucosa in certain proportion after oral administration, and reached the colonization peak at 6 days after oral administration, and 15.23 percent in duodenum, jejunum, ileum and cecum at 7 days after oral administration. 24.19 there are 48.57 and 40.07. The results showed that Proteus mirabilis ompA could be stably expressed on the surface of Lactococcus lactis, and the recombinant bacteria had good colonization ability in mouse intestine, and the colonization rule was jejunum duodenum of ileum caecum. This provides the experimental basis for the study of Lactococcus lactis as an oral vaccine of Proteus mirabilis and its effect on the intestinal immune mechanism of mice. The effects of Taishanpine Pollen Polysaccharide (TPPPS) as adjuvant on immune function in mice were investigated. The results showed that the recombinant Lactococcus lactis oral vaccine could induce specific IgA and IgG,IL-2,. The production of IL-4 and IFN- 緯 promoted the proliferation of T lymphocytes, and the immunized mice could significantly resist the colonization of Proteus mirabilis in the intestine. In addition, TPPPS adjuvant could induce higher levels of IgA,IgG,IL-2,IL-4 and IFN- 緯, as well as T lymphocyte proliferation, compared with recombinant lactococcus lactis oral vaccine. To sum up, Lactococcus lactis combined with TPPPS adjuvant to prepare oral vaccine provides a new method to prevent Proteus mirabilis infection.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.26
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