1、 補體補體C3dC3d增強(qiáng)偽狂犬病毒增強(qiáng)偽狂犬病毒gCgC基因免疫基因免疫原性的研究原性的研究匯報提綱匯報提綱文獻(xiàn)綜述文獻(xiàn)綜述研究的目的、意義研究的目的、意義研究內(nèi)容研究內(nèi)容結(jié)果與分析結(jié)果與分析結(jié)論結(jié)論補體C3dC3d的分子生物學(xué)特性補體C3d晶體格結(jié)構(gòu)補體受體CR2晶體格結(jié)構(gòu)補體受體CR2與C3d結(jié)合的晶體格結(jié)構(gòu)抗原和補體C3d復(fù)合物與B細(xì)胞表面受體結(jié)合示意圖 補體C3d的作用機(jī)理偽狂犬病毒偽狂犬病毒gCgC基因的結(jié)構(gòu)與功基因的結(jié)構(gòu)與功能能 糖蛋白介導(dǎo)著病毒與細(xì)胞相互作用，同時也是動物機(jī)體的免疫系統(tǒng)識別的主要靶抗原。 偽狂犬病毒病毒粒子結(jié)構(gòu)ProteinProteinProtectionPr

2、otectionNT-CNT-CNT+CNT+CProlif.Prolif.CTL(MHCCTL(MHC-I restr.)-I restr.)CTL(LAK)CTL(LAK)MiceMicePigsPigsgBgB+ + + + + +? ?+ +gCgC+ + + + + + + +gDgD+ + + + + +? ?- -gEgE+ +? ?- -+ +- - -? ?gGgG- - - - - - -? ?gHgH? ? ? ? ?+ +? ? ?gIgI? ? ?- -? ?- - -? ?gKgK? ? ? ? ? ? ? ?gLgL? ? ? ? ? ? ? ?gMgM? ? ?

3、 ? ? ? ? ?gNgN ？ ？ ？ ？ ？ ？PrV 11PrV 11種糖蛋白在免疫中的作用種糖蛋白在免疫中的作用研究內(nèi)容：研究內(nèi)容：1.1.豬、鼠、羊補體C3dC3d分子cDNAcDNA的克隆與序列分析2.2.補體C3d或受體結(jié)合功能區(qū)M28對gC基因核酸疫苗的免疫增強(qiáng)作用3.3.豬源補體C3d的分子免疫增強(qiáng)作用及小鼠動物模型的建立試驗技術(shù)路線圖試驗技術(shù)路線圖RT-PCRRT-PCR擴(kuò)增補體C3dC3d分子重組質(zhì)粒的構(gòu)建動物免疫試驗體液免疫 細(xì)胞免疫結(jié)果與分析豬、鼠、羊補體豬、鼠、羊補體C3d分子的克隆分子的克隆 UL IR Us TR小鼠肝細(xì)胞總RNA的電泳結(jié)果 C3d cDNA的R

4、T-PCR擴(kuò)增不同物種間C3d氨基酸同源性的比較 鼠/羊補體C3d同源性81%，鼠/豬補體C3d同源性82% 100cattle_c3d 100chicken_c3d 100golden_hamster_c 100human_c3d 100mouse_c3d 100ovis_aries_c3d 100pig_c3d 100sheep_c3dTTTTTTTTPPPPPPPPSSSSASSSGGGGGGGGCCCCCCCCGGGGGGGGEEEEEEKEQQQQQQQPNNNNHNTNMMMMMMMMIIIIIIIIGGGGGGGGMMMMMMMMTTTTTTTTPPPPPPPPTTTTTTTTV

5、VVVVVVVIIIIIIIIAAAAAAAAVVVVVVVVHHHHHHHHYYYYYYYYLLLLLLLLDDDDDDDDSSQEQSSSTTTTTTTTDMEEEEEEQQQQQQQQWWWWWWWWEEEEGEEEKTKKKKKKFFFFFFFFGGGGGGGGLILLIMLVENQEEEEEKRKKKKKKRRRRRRRRQTQQQQQQEEEGEEEESASAAAAALILLLLLLEEEEEEEELLLLLLLLIIIIIIIIRKKKKRKRKKKKKKKKGGGGGGGGYYYYYYYYTTTTTTTTQQQQQQQQQQQQQQQQLLLLLLLLAAAAAAAAFYFFF

6、FFFRRKRKRRRQKQQQQQQKEPPPKKKSDSSSSNSSGFSSSSSASAAAAAAYFYFYYFYAAAAAAAAAAAAAAAAFFFFFFFFQTNVNQQQYTNKNNDNRRRRRRRRPPPAPPLPPSPPPPSPSSSSSSSSTTTTTTTTWWWWWWWWLLLLLLLLTTTTTTTTAAAAAAAAYYYYYYYYVVVVVVVVVAVVVVVVKKKKKKKKVVVVVVVVFFFFFFFFAASSSAAALMLLLLMLAAAAASASAIAVATAANNNNNNNNLMLLLLLLIVIIIIIIADAAAAAAIIIIIIIIDKDDDDDDS

7、PSSSSSSKEQQHRQRDVVVVDVDLVLLLLLLCCCCCCCCEGGGGEGETAAAATATVIVVVVVVKKKKKKKK 200cattle_c3d 199chicken_c3d 200golden_hamster_c 200human_c3d 200mouse_c3d 200ovis_aries_c3d 200pig_c3d 200sheep_c3dWWWWWWWWLLLLLLLLIIIIIIIILLLLLLLLEEEEEEEEKKKKKKKKQQQQQQQQKQKKKKKKPPPPPPPPDDDDDDDDGGGGGGGGILVVVIVIFFFFFFFFQQQQQQEQ

8、EEEEEEEEDDDDDDNDGAGAGGGGPPPPPPPPVVVVVVVVIIIIIIIIHHHHHHHHQKQQQQQQEEEEEEEEMMMMMMMMIVIIIIIIGGGGGGGGGGGGGGGGFYFLFFFFRHRRRKKKDGNNNDNDT.ANATTARAKNKREREEEEEEEEKPAKAKKKDSDDDDDDVVVMVVVVSSSASSSSLLLLLLLLTTTTTTTTAAAAAAAAFFFFFFFFVVVVVVVVLLLLLLLLISIIIIIIAAASAAAALLLLLLLLHQQQQHQHEEEEEEEEASAAAAAAKQRKRKKKDKEDDDDGIIII

9、IIIICCCCCCCCEKEEEEEEANGEGAPAQYQQQQQQVVIVVVVVNKNNNNNNSSSSSSSSLLLLLLLLGDPPPGLGRGGGGPRPSSSSSSSSIIIIIIIIAANTNTNTKKKKKKKKAAAAAAAAGSGGGGRGDDEDEDDNFYYFYFFFLLILILLLESEEEEAENRAAANDNHKSNSHYHYYYYYYYYRQLMMRLRESNNNEEELLLLLLLLRTQQQRKRRRRRRRRRPPPSPPPPYYYYYYYYTTTTTTTTVVVVVVVVAAAAAAAAILIIIIIIATAAAAAAASAGGAGAYYYYYYYY

10、AAAAAAAALLLLLLLLAAAAAAAALLLQLLLLLTMMMLSLGGGGNGDGKKKRKKKKLLLLLLLLENEKEEDEGSEGEDEDDEPPPDPD 299cattle_c3d 295chicken_c3d 298golden_hamster_c 298human_c3d 299mouse_c3d 298ovis_aries_c3d 296pig_c3d 289sheep_c3dR.YLYRFRL.LLLLLLTKSNGTNTKVKKKKKKFLFFFFLFLMLLLLLLNKKTNNSNTFTTTTTTASAAAAAAKKTKKKKKEDEDDEEEKGRKRKR

11、KNTNNNNNNRHRRRRRRWWWWWWWWEAEEEEEEEEEDEEEEPRPPPPPPNNGGDNGNQAQKQKQKKHKQQKKKLTLLLLLLYYYYYYYYNNNNNNNNVIVVVVVVEEEEEEEEAGAAAAAATTTTTTTTSSSSSSSSYYYYYYYYAAAAAAAALLLLLLLLLLLLLLLLAAAAAAAALLLLLLLLLLLLLLLLAQLQLAVARMLLLRVRKEKKKKKKDKDDDDDDYAFFFFFFDEDDDDDDTLSFSTSTTTVVVVVVPGPPPPPPPPPPPPPPVVVVVVIVVVVVVVVVRRRRRRRRWWW

12、WWWWWLLLLLLLLNANNNNNNEQEEEEEEQQQQQQQQRNRRRRRRYYYYYYYYYFYYYYYYGGGGGGGGGGGGGGGGGGGGGGGGYYYYYYYYGGGGGGGGSSSSSSSSTTTTTTTTQQQQQQQQAAAAAAAATTTTTTTTFIFFFFFFMLMMMMMMVVVVVVVVFFFFFFFFQQQQQQQQAAAAAAAALLLLLLLLAAAAAAAAQQQQQQQQYYYYYYYYQHQQQQQQKVTKTKKKDADDDDDDVLVAVVVVPPPPPPPPDRDDDDDDHQHHHHHHKLKQKKKKEEDEDEDELLLLLLL

13、LNNNNNNNNLLMLMLLLDDDDDDDVVVVVVVSSSSSSSIVFLFIIQLHQHHHLLLLLLLPPPPPPPSRSSSSRRRRRRNS 兩種不同形式的系統(tǒng)發(fā)育進(jìn)化兩種不同形式的系統(tǒng)發(fā)育進(jìn)化樹樹 mouse_c3dgolden_hamster_chuman_c3dpig_c3dsheep_c3dovis_aries_c3dcattle_c3dchicken_c3d0.05mouse_c3dmouse_c3golden_hamster_chuman_c3pig_c3dpig_c3sheep_c3dsheep_c3cattle_c3dchicken_c3dchicken_c

14、30.05鼠、豬、羊補體C3d蛋白信號肽分析 鼠、豬、羊補體C3d均無信號肽 鼠、豬、羊的受體結(jié)合功能區(qū) 重組表達(dá)質(zhì)粒的結(jié)構(gòu)示意圖 重組質(zhì)粒的間接免疫熒光試驗A: pcD NA3.1+; B: sgC; C: sgC-C3d3; D: sgC-M284ABDCWestern blotting 分析分析融合蛋白表達(dá)融合蛋白表達(dá) Western blot Western blot 檢測pcDNA3.1+ pcDNA3.1+ 、sgCsgC、 sgC-C3d3sgC-C3d3、sgC-sC3d3sgC-sC3d3和 sgC-M284 sgC-M284 在PK-15 PK-15 細(xì)胞中的表達(dá)不同免疫組

15、誘導(dǎo)的抗不同免疫組誘導(dǎo)的抗gC ELISA抗體檢抗體檢測測 鼠源C3d作用豬源C3d作用免疫組不同階段誘導(dǎo)的中和抗體滴免疫組不同階段誘導(dǎo)的中和抗體滴度度 鼠源補體C3d豬源補體C3d各免疫組誘導(dǎo)的淋巴細(xì)胞刺激指各免疫組誘導(dǎo)的淋巴細(xì)胞刺激指數(shù)數(shù) 鼠源補體c3d作用豬源補體c3d作用 免疫組脾細(xì)胞IL-4、 IFN-水平檢測 鼠源補體c3d免疫增強(qiáng)作用鼠源補體c3d免疫增強(qiáng)作用攻毒后小鼠的存活情況 鼠源補體C3d豬源補體C3d攻毒后各免疫組小鼠的體重變化情況 鼠源補體c3d作用豬源補體c3d作用結(jié)論結(jié)論. .克隆了豬、鼠、羊的補體C3dC3d分子，其中羊補體C3dC3d分子的克隆為首次報道。 2.

16、2. 證明gCgC基因與C3dC3d融合能夠顯著增強(qiáng)gCgC基因常規(guī)DNADNA疫苗所誘發(fā)的體液免疫水平，調(diào)節(jié)Th1/Th2Th1/Th2免疫反應(yīng)平衡，提供更強(qiáng)的免疫保護(hù)作用。3. 3. 補體C3d的受體結(jié)合功能區(qū)M28可以增強(qiáng)gC基因常規(guī)DNA疫苗所誘發(fā)的ELISA與中和抗體水平，但是較全長C3d的免疫增強(qiáng)作用略差。4. 豬源補體C3d同樣可以顯著增強(qiáng)gC基因常規(guī)DNA疫苗抗體水平，提供更強(qiáng)的免疫保護(hù)作用。5.建立了小鼠模型，研究豬源補體C3d分子免疫佐劑作用。1.Tiezhu Tong, Huiying Fan, Yadi Tan, Shaobo Xiao, Jieyu Ling, Hua

17、nchun Chen, Aizhen Guo C3d enhanced DNA vaccination induced humoral immune response to glycoprotein gC of pseudorabies virus Biochemical and Biophysical Research Communications（2004IF: 2.904）2. Tiezhu Tong, Huiying Fan, Yadi Tan, Shaobo Xiao, Huanchun Chen, Aizhen Guo Immunogenicity of a Pseudorabie

18、s virus gCC3d fusionprotein containing a porcine homolog of C3d Vaccine (submitted)3.Tiezhu Tong, Huiying Fan, Yadi Tan, Huanchun Chen, Aizhen Guo C3d enhancement of gC glycoprotein immune response against pseudorabies virus 19th International Pig Veterinary Congress碩士期間發(fā)表文章：碩士期間發(fā)表文章：4.Huiying Fan, Chunmei,Ju, Tiezhu Tong, Hongliang Huang, Jiangqiang Lv, Huanchun Chen Immunogenicity of E