【摘要】：大腸桿菌(Escherichia coli),是一種重要的人畜共患病病原菌。該菌在環(huán)境中廣泛存在,部分菌株具有較強(qiáng)的致病性,菌蛻(BGs)是革蘭陰性菌被噬菌體PhiX174裂解基因E裂解后形成的完整細(xì)菌空殼,無(wú)細(xì)胞內(nèi)容物。除可以作為疫苗外,還可作為佐劑、外源蛋白抗原、生物活性物質(zhì)和DNA疫苗載體等多種功能。僅通過(guò)基因E裂解制備菌蛻,還可能具有病原島或抗生素抗性基因水平轉(zhuǎn)移等風(fēng)險(xiǎn)。因此,作為疫苗或疫苗載體的菌蛻應(yīng)當(dāng)更為安全。為了盡量消除裂解后細(xì)菌的活力并清除殘留的遺傳物質(zhì),本研究又將輔助致死基因SNA引入裂解表達(dá)系統(tǒng)中,并構(gòu)建了新型溫控表達(dá)質(zhì)粒pBVE-CIP-SNA(pBVES),成功導(dǎo)入牛源大腸桿菌O14野生株,且進(jìn)行反復(fù)多次溶菌動(dòng)力學(xué)試驗(yàn),質(zhì)粒型大腸桿菌菌蛻裂解率高達(dá)99.65%,用透射電鏡進(jìn)行觀(guān)察,與正常狀態(tài)大腸相比菌蛻內(nèi)容物外排徹底,形態(tài)及表面結(jié)構(gòu)完整。為驗(yàn)證菌蛻免疫原性,研究設(shè)立菌蛻組、滅活組、滅活佐劑組為試驗(yàn)組,對(duì)照組為PBS組,通過(guò)測(cè)定不同免疫指標(biāo)來(lái)比較四組的免疫原性。結(jié)果顯示:隨著免疫次數(shù)增加,試驗(yàn)組血清IgG含量明顯高于對(duì)照組,小鼠淋巴細(xì)胞亞群試驗(yàn)結(jié)果表明菌蛻能刺激小鼠機(jī)體產(chǎn)生Th1型細(xì)胞免疫應(yīng)答,而小鼠主要細(xì)胞因子檢測(cè)結(jié)果表明菌蛻組的IFN-γ和IL-12分泌水平明顯高于其他試驗(yàn)組和對(duì)照組,且試驗(yàn)組均高于對(duì)照組水平,但I(xiàn)L-4試驗(yàn)組的分泌水平與對(duì)照組差異不明顯。攻毒保護(hù)結(jié)果顯示,菌蛻組對(duì)小鼠機(jī)體保護(hù)率可達(dá)到70%。臨床癥狀表明,在攻毒后,菌蛻組小鼠狀態(tài)優(yōu)于其他免疫組。綜上所述,質(zhì)粒型大腸桿菌菌蛻具有較好的免疫原性,研究結(jié)果為新型疫苗的研究提供依據(jù),具有廣闊的應(yīng)用前景。
[Abstract]:E. coli (Escherichia coli), is an important zoonotic pathogen. Some strains have strong pathogenicity. Slough (BGs) is an intact bacterial shell formed by E cleavage of bacteriophage PhiX174 cleavage gene, and has no cell contents. It can also be used as adjuvant, foreign protein antigen, bioactive substance and DNA vaccine carrier. The preparation of mycelium slough by gene E cleavage may also have the risk of pathogenetic island or horizontal transfer of antibiotic resistant gene. Therefore, it should be safer to use as vaccine or vaccine carrier. In order to eliminate the viability of the bacteria and remove the residual genetic material, the co-lethal gene SNA was introduced into the lytic expression system, and a new thermo-controlled expression plasmid pBVE-CIP-SNA (pBVES), was constructed. The wild strain of Escherichia coli O14 from cattle was successfully introduced, and repeated bacteriolysis kinetics tests were carried out. The slough cleavage rate of plasmid type Escherichia coli was up to 99.65, which was observed by transmission electron microscope (TEM). Compared with the normal large intestine, the fungicidal shedding contents were completely discharged, and the morphology and surface structure were complete. In order to verify the immunogenicity of mycelium slough, the study established the mycelium slough group, the inactivated adjuvant group as the experimental group and the control group as the PBS group. The immunogenicity of the four groups was compared by measuring different immune indexes. The results showed that the level of serum IgG in the experimental group was significantly higher than that in the control group with the increase of immunization times. The results of lymphocyte subgroup test showed that the mycelium could stimulate the Th1 type cellular immune response in mice. The levels of IFN- 緯 and IL-12 in the mycelia group were significantly higher than those in the other test groups and control groups, and the levels in the test group were higher than those in the control group. However, the secretory level of IL-4 test group was not significantly different from that of control group. The results showed that the protective rate of mycelium slough group could reach 70%. The clinical symptoms showed that the state of the mice in the mycelia group was better than that in the other immune groups. To sum up, plasmid Escherichia coli slough has good immunogenicity, the research results provide the basis for the study of new vaccine, and have a broad application prospect.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.61

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