重組NDV HN基因減毒雞白痢沙門(mén)菌的構(gòu)建及其特性研究
發(fā)布時(shí)間：2018-09-03 10:24
【摘要】：新城疫是由新城疫病毒引發(fā)的一種急性和高度接觸性禽類(lèi)疾病,對(duì)世界養(yǎng)禽業(yè)造成了毀滅性的破壞,世界動(dòng)物衛(wèi)生組織將其列入必須進(jìn)行通報(bào)的動(dòng)物疾病之一。近年來(lái),由于傳統(tǒng)疫苗對(duì)新城疫預(yù)防的種種缺陷,研制一種新型基因工程疫苗來(lái)預(yù)防和控制新城疫勢(shì)在必行。雞白痢沙門(mén)菌(S.Pullorum)是目前危害養(yǎng)雞業(yè)的重要細(xì)菌之一,藥物控制該菌易造成生物安全問(wèn)題,傳統(tǒng)疫苗免疫力不高,因此,將減毒沙門(mén)菌作為預(yù)防細(xì)菌疾病的新型活疫苗是當(dāng)下控制雞白痢的首選途徑。本試驗(yàn)在減毒S.Pullorum C79-13?crp的基礎(chǔ)上,進(jìn)一步缺失asd基因,利用其質(zhì)粒平衡致死系統(tǒng)構(gòu)建重組菌株C79-13Δcrp?asd(p YA-HN),并對(duì)其生物學(xué)及免疫學(xué)特性進(jìn)行研究。1.減毒S.Pullorum C79-13?crp?asd株的構(gòu)建及其生物學(xué)特性本研究以S.Pullorum C79-13?crp作為受體菌、大腸桿菌χ7213(p REΔasd)作為供體菌,將二者進(jìn)行接合轉(zhuǎn)移,并通過(guò)“兩步法”篩選asd基因缺失株,PCR鑒定結(jié)果及測(cè)序結(jié)果顯示,成功構(gòu)建S.Pullorum缺失株C79-13?crp?asd。生物學(xué)特性結(jié)果表明,該菌在DAP陰性環(huán)境中存活的能力喪失,同時(shí)也失去了利用麥芽糖、葡萄糖、甘露醇和鼠李糖等碳源的能力;其血清型與野生株C79-13一致,生長(zhǎng)速度減慢;能夠在體外穩(wěn)定遺傳asd缺失基因;雛雞口服攻毒試驗(yàn)結(jié)果顯示重組菌株LD50較野生株C79-13升高約3.1×104倍。2.重組NDV HN減毒S.Pullorum的構(gòu)建及其生物學(xué)特性本試驗(yàn)將PCR擴(kuò)增出的HN基因片段克隆至無(wú)抗性原核表達(dá)載體p YA3493,得到重組表達(dá)質(zhì)粒p YA-HN,進(jìn)而將經(jīng)PCR及酶切鑒定正確的重組質(zhì)粒導(dǎo)入C79-13?crp?asd中構(gòu)建重組菌株C79-13Δcrp?asd(p YA-HN)。該重組菌株的生化特性與生長(zhǎng)特性與C79-13?crp?asd基本保持一致;NDV HN基因能夠在體外穩(wěn)定遺傳;SDS-PAGE結(jié)果顯示HN基因在重組菌中得到了表達(dá),Western blot結(jié)果表明表達(dá)蛋白具有良好的免疫原性;重組菌毒力較野生株C79-13升高約3.4×104倍;定植試驗(yàn)結(jié)果顯示,重組菌對(duì)雞體具有較好的侵襲力。3.重組菌株C79-13Δcrp?asd(p YA-HN)的免疫學(xué)特性研究重組菌株口服免疫雛雞,對(duì)其免疫學(xué)特性進(jìn)行研究。結(jié)果表明,對(duì)雞只體重的增加與對(duì)照組相比差異不顯著(P0.05),能顯著刺激雞體免疫器官指數(shù)的增加(P0.05);血清HI抗體測(cè)定結(jié)果顯示,HI抗體水平于免疫后21d時(shí)達(dá)到最大值(log2=5.3);重組菌免疫后第21d免疫血清中沙門(mén)菌Ig G抗體水平顯著高于對(duì)照組(P0.05);在重組菌株免疫雞腸道沖洗液中檢測(cè)到較PBS組高水平的分泌型Ig A(log2=3.56);免疫組所分離的淋巴細(xì)胞經(jīng)體外刺激后其SI在免疫第21d達(dá)到最大值,即1.698,與PBS組相比差異顯著(P0.05);免疫保護(hù)試驗(yàn)結(jié)果顯示,重組菌免疫組可能100%抵抗沙門(mén)菌野生株的感染,對(duì)NDV F48E8的攻擊有60%的保護(hù)率。綜上所述,重組菌免疫后在一定時(shí)期內(nèi)能夠刺激機(jī)體產(chǎn)生有效的體液、黏膜和細(xì)胞免疫應(yīng)答反應(yīng),且對(duì)S.Pullorum及NDV野毒株的感染具有保護(hù)效果。
[Abstract]:Newcastle disease (NDV) is an acute and highly contact avian disease caused by Newcastle disease virus (NDV), which has caused devastating damage to the world poultry industry, and has been listed as one of the animal diseases that must be notified by the World Organization of Animal Health (OIE). In recent years, it is imperative to develop a new genetic engineering vaccine to prevent and control Newcastle disease because of the defects of traditional vaccines. Salmonella pullorum (S.Pullorum) is one of the most important bacteria that harm the chicken industry at present. Drug control of Salmonella pullorum is easy to cause biosafety problems, and the immunity of traditional vaccine is not high. Attenuated Salmonella as a new live vaccine to prevent bacterial disease is the first choice to control chicken pullorum. The recombinant strain C79-13 螖 crp?asd (p YA-HN) was constructed on the basis of attenuated S.Pullorum C79-13?crp and further deletion of asd gene. The biological and immunological characteristics of C79-13 螖 crp?asd (p YA-HN) were studied. Construction and Biological characteristics of attenuated S.Pullorum C79-13?crp?asd strain. In this study, S.Pullorum C79-13?crp was used as receptor strain and 蠂 7213 (p RE 螖 asd (Escherichia coli) as donor strain, and the two strains were conjugated and transferred. The results of identification and sequencing of asd gene deletion strain by "two-step method" showed that the S.Pullorum deletion strain C79-13 was successfully constructed. The biological characteristics showed that the strain lost its ability to survive in DAP negative environment and lost the ability to utilize carbon sources such as maltose, glucose, mannitol and rhamnose, and its serotype was consistent with that of wild strain C79-13, and its growth rate slowed down. The results showed that the LD50 of the recombinant strain was 3.1 脳 10 ~ 4 times higher than that of the wild strain C79-13. Construction and Biological characteristics of attenuated S.Pullorum of Recombinant NDV HN the HN gene fragment amplified by PCR was cloned into the non-resistant prokaryotic expression vector p YA3493, to obtain the recombinant expression plasmid p YA-HN, which was identified correctly by PCR and restriction endonuclease digestion. The recombinant strain C79-13 螖 crp?asd (p YA-HN) was constructed by introducing the plasmid into C79-13?crp?asd. The biochemical and growth characteristics of the recombinant strain were basically consistent with that of C79-13?crp?asd. The results of SDS-PAGE showed that the HN gene was expressed in the recombinant strain. The results showed that the expressed protein had good immunogenicity. The virulence of the recombinant strain was about 3.4 脳 104 times higher than that of the wild strain C79-13, and the results of colonization test showed that the recombinant strain had a better invasiveness to chicken body. Immunological characteristics of Recombinant strain C79-13 螖 crp?asd (p YA-HN) the immunological characteristics of recombinant strain C79-13 螖 crp?asd (p YA-HN) were studied. The results show that The increase in body weight of chickens was not significantly different from that of the control group (P0.05), but it could significantly stimulate the increase of body immune organ index (P0.05), the level of serum HI antibody reached the maximum value (log2=5.3) at 21 days after immunization, and the recombinant bacteria were immunized. The antibody level of Salmonella Ig G in serum was significantly higher than that in the control group on the 21st day after immunization (P0.05), the secretory Ig A (log2=3.56 was detected in the intestinal lavage fluid of the recombinant strain immunized chicken, and the lymphocytes isolated from the immunized group were stimulated in vitro. The SI reached the maximum value on the 21st day after immunization. The results of immunoprotection test showed that 100% of the recombinant bacteria immunized group could resist the infection of Salmonella wild-strain and the protective rate of NDV F48E8 was 60%. To sum up, the recombinant bacteria can stimulate the body to produce effective humoral, mucosal and cellular immune responses in a certain period after immunization, and have protective effect on the infection of S.Pullorum and NDV wild strains.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.6
本文編號(hào)：2219704
[Abstract]:Newcastle disease (NDV) is an acute and highly contact avian disease caused by Newcastle disease virus (NDV), which has caused devastating damage to the world poultry industry, and has been listed as one of the animal diseases that must be notified by the World Organization of Animal Health (OIE). In recent years, it is imperative to develop a new genetic engineering vaccine to prevent and control Newcastle disease because of the defects of traditional vaccines. Salmonella pullorum (S.Pullorum) is one of the most important bacteria that harm the chicken industry at present. Drug control of Salmonella pullorum is easy to cause biosafety problems, and the immunity of traditional vaccine is not high. Attenuated Salmonella as a new live vaccine to prevent bacterial disease is the first choice to control chicken pullorum. The recombinant strain C79-13 螖 crp?asd (p YA-HN) was constructed on the basis of attenuated S.Pullorum C79-13?crp and further deletion of asd gene. The biological and immunological characteristics of C79-13 螖 crp?asd (p YA-HN) were studied. Construction and Biological characteristics of attenuated S.Pullorum C79-13?crp?asd strain. In this study, S.Pullorum C79-13?crp was used as receptor strain and 蠂 7213 (p RE 螖 asd (Escherichia coli) as donor strain, and the two strains were conjugated and transferred. The results of identification and sequencing of asd gene deletion strain by "two-step method" showed that the S.Pullorum deletion strain C79-13 was successfully constructed. The biological characteristics showed that the strain lost its ability to survive in DAP negative environment and lost the ability to utilize carbon sources such as maltose, glucose, mannitol and rhamnose, and its serotype was consistent with that of wild strain C79-13, and its growth rate slowed down. The results showed that the LD50 of the recombinant strain was 3.1 脳 10 ~ 4 times higher than that of the wild strain C79-13. Construction and Biological characteristics of attenuated S.Pullorum of Recombinant NDV HN the HN gene fragment amplified by PCR was cloned into the non-resistant prokaryotic expression vector p YA3493, to obtain the recombinant expression plasmid p YA-HN, which was identified correctly by PCR and restriction endonuclease digestion. The recombinant strain C79-13 螖 crp?asd (p YA-HN) was constructed by introducing the plasmid into C79-13?crp?asd. The biochemical and growth characteristics of the recombinant strain were basically consistent with that of C79-13?crp?asd. The results of SDS-PAGE showed that the HN gene was expressed in the recombinant strain. The results showed that the expressed protein had good immunogenicity. The virulence of the recombinant strain was about 3.4 脳 104 times higher than that of the wild strain C79-13, and the results of colonization test showed that the recombinant strain had a better invasiveness to chicken body. Immunological characteristics of Recombinant strain C79-13 螖 crp?asd (p YA-HN) the immunological characteristics of recombinant strain C79-13 螖 crp?asd (p YA-HN) were studied. The results show that The increase in body weight of chickens was not significantly different from that of the control group (P0.05), but it could significantly stimulate the increase of body immune organ index (P0.05), the level of serum HI antibody reached the maximum value (log2=5.3) at 21 days after immunization, and the recombinant bacteria were immunized. The antibody level of Salmonella Ig G in serum was significantly higher than that in the control group on the 21st day after immunization (P0.05), the secretory Ig A (log2=3.56 was detected in the intestinal lavage fluid of the recombinant strain immunized chicken, and the lymphocytes isolated from the immunized group were stimulated in vitro. The SI reached the maximum value on the 21st day after immunization. The results of immunoprotection test showed that 100% of the recombinant bacteria immunized group could resist the infection of Salmonella wild-strain and the protective rate of NDV F48E8 was 60%. To sum up, the recombinant bacteria can stimulate the body to produce effective humoral, mucosal and cellular immune responses in a certain period after immunization, and have protective effect on the infection of S.Pullorum and NDV wild strains.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.6
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 武發(fā)菊;劉學(xué)榮;尚勇良;安芳蘭;董文教;宋玉霞;董金杰;陳苗苗;牟克斌;;新城疫疫苗的研究進(jìn)展[J];中國(guó)畜牧獸醫(yī);2011年10期
2 梅春升;周世霞;馬立保;;新城疫疫苗及其應(yīng)用研究進(jìn)展[J];國(guó)外畜牧學(xué)(豬與禽);2006年06期
3 劉一塵;張春杰;程安春;程相朝;李銀聚;吳庭才;易明林;;新城疫病毒HN基因在畢赤酵母中的表達(dá)及其表達(dá)產(chǎn)物的免疫原性[J];中國(guó)獸醫(yī)科學(xué);2008年10期
4 張永宏;;新城疫的流行特點(diǎn)與發(fā)病原因分析[J];中國(guó)畜禽種業(yè);2014年12期
相關(guān)博士學(xué)位論文 前1條
1 丁軍濤;表達(dá)Tsol18抗原的口服減毒鼠傷寒沙門(mén)氏菌重組活載體疫苗的構(gòu)建及其免疫學(xué)研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2008年
相關(guān)碩士學(xué)位論文 前1條
1 劉新軍;表達(dá)布氏桿菌sodc基因的重組沙門(mén)氏菌的構(gòu)建及不同種布氏桿菌鑒別PCR檢測(cè)方法的建立[D];華中農(nóng)業(yè)大學(xué);2010年
，本文編號(hào)：2219704
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2219704.html
最近更新
教材專(zhuān)著
