【摘要】：目的利用成功構(gòu)建好的含有日本血吸蟲重組信號蛋白14-3-3(rSj14-3-3)和重組Mr26000谷胱甘肽S轉(zhuǎn)移酶(rSj26GST)編碼基因的菌株E.coli BL21/pET28a,誘導(dǎo)表達rSj14-3-3和rSj26GST這兩種重組蛋白,并利用其免疫小鼠,觀察日本血吸蟲rSj14-3-3疫苗和rSj26GST疫苗聯(lián)合免疫對小鼠的保護作用,同時觀察日本血吸蟲rSj14-3-3和rSj26GST疫苗聯(lián)合免疫對小鼠抗病免疫效應(yīng)的研究。 方法分別復(fù)蘇本實驗室-80℃低溫保種的含有rSj14-3-3和rSj26GST編碼基因的菌株E.coliBL21/pET28a,將其均勻涂布于LB/Kana/IPTG/ X-gal平板,培養(yǎng)、收集細菌、超聲碎菌、分離、親和層析法純化重組蛋白,分別取5μl進行SDS-PAGE ,觀察純化結(jié)果,采用Bradford法測定重組蛋白rSj14-3-3和rSj26GST的濃度。將100只6～8周齡BALB/c雌性小鼠隨機分為A、B、C、D、E5組,每組20只,將兩種純化后的蛋白分別皮下免疫BALB/c小鼠。A組(感染對照組),于第0周每鼠經(jīng)背部皮下多點注射50μl PBS及等體積福氏完全佐劑(CFA),于第2及4周每鼠皮下多點加強注射50μl PBS加等體積福氏不完全佐劑(IFA)。B組(rSj14-3-3組),每鼠于第0、2、4周經(jīng)皮下注射100μg(50μl)rSj14-3-3。C組(rSj26 GST組),每鼠于第0、2、4周經(jīng)皮下注射100μg(50μl)rSj26 GST。D組(rSj14-3-3 + rSj26 GST聯(lián)合免疫組),于第0、2、4周經(jīng)背部皮下多點注射rSj14-3-3和rSj26 GST各50μg(25μl)。上述各組分別于末次免疫2周后每鼠經(jīng)腹部皮膚感染日本血吸蟲尾蚴(30±2)條。E組(對照組)未作任何處理。于感染45d后剖殺全部小鼠,眼眶取血,用于血清纖維化指標檢測;計數(shù)成蟲及肝內(nèi)蟲卵,計算各組間的減蟲率和減卵率;將小鼠肝組織做成切片進行HE染色,測量各組單卵肉芽腫大小。 結(jié)果成功誘導(dǎo)表達rSj14-3-3和rSj26GST這兩種重組蛋白,并對其進行純化,經(jīng)鑒定純化結(jié)果較好。聯(lián)合免疫組的減蟲率為38.4%,顯著高于rSj14-3-3(19.8%)和rSj26GST組(21.6%)(P值均0.01)。聯(lián)合免疫組以及rSj14-3-3、rSj26GST組減卵率分別為48.8%、35.3%、41.4%;聯(lián)合免疫組及rSj14-3-3和rSj26GST組,肝組織中每條雌蟲平均產(chǎn)卵數(shù)顯著低于感染對照組(P值均0.01)。聯(lián)合疫苗組小鼠蟲卵肉芽腫的直徑為(173.9±35.0um),顯著小于感染對照組的(267.7±28.6um)(P值0.01)。檢測各組的透明質(zhì)酸和層黏連蛋白水平,聯(lián)合免疫疫苗組顯著低于感染對照組(P值0.01),他們均高于正常對照組。 結(jié)論成功誘導(dǎo)表達rSj14-3-3和rSj26GST重組蛋白,并成功獲得他們純化后的重組蛋白。聯(lián)合免疫組的保護作用優(yōu)于rSj14-3-3和rSj26GST單獨免疫組。聯(lián)合免疫疫苗具有一定的抗蟲卵肉芽腫及抗肝纖維化作用。
[Abstract]:Objective to construct a recombinant Schistosoma japonicum (Schistosoma japonicum) strain E.coli BL21/pET28a, which contains recombinant signal protein (rSj14-3-3) and recombinant glutathione S transferase (rSj26GST) gene of Schistosoma japonicum. The recombinant proteins rSj14-3-3 and rSj26GST were induced and immunized in mice. The protective effects of combined immunization with rSj14-3-3 vaccine and rSj26GST vaccine against Schistosoma japonicum were observed. At the same time, the effect of combined immunization with rSj14-3-3 and rSj26GST vaccine of Schistosoma japonicum on anti-disease immunity in mice was observed. Methods E.coli BL21 / pET28a, which contained rSj14-3-3 and rSj26GST coding genes, was revived at-80 鈩，

本文編號：2472278