口蹄疫新型疫苗研究
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摘要
口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouth diseasevirus,FMDV)引起的,偶蹄动物共患的烈性、急性、接触性传染病。尽管该病的死亡率低,但由于该病传染性极强,能迅速形成大范围流行,而导致发病地区、乃至该国的畜产品出口贸易停止,从而造成巨大的经济损失。免疫接种是预防和控制该病的主要措施,目前用于预防FMD的主要疫苗是传统的弱毒苗和灭活苗,虽然这些传统疫苗能够提供较好的保护,但存在制备费用高、潜在的散毒危险等缺点,使得这两种疫苗的使用一直都存在着一定的争议。因此,研制更安全、高效、廉价的新型疫苗成为越来越多研究者关注的焦点。
鉴于此,本研究对FMDV新型疫苗设计进行了新的探索,以构建了伪狂犬病毒(PRV)为载体的重组PRV-FMDV新型基因工程疫苗,以西门利克森林病毒(SFV)为复制子的“自杀性”DNA疫苗和以杆状病毒为载体的重组病毒疫苗,并对构建的三种候选疫苗进行了免疫效力研究;克隆并表达了猪源髓细胞分化因子MyD88,并探讨了其作为分子佐剂在小鼠体内对FMDV DNA疫苗免疫效果的影响。主要研究内容如下:
1、共表达0型口蹄疫衣壳前体蛋白P1及其多抗原合成表位FHG的重组伪狂犬病毒FHG/P1/PRV的构建和免疫原性研究
根据已公布的O型口蹄疫抗原表位设计合成了多抗原表位肽基因fhg,将其插入到中间转移载体pIECMV中,获得重组中间转移质粒pIEFHG,将pIEFHG与本室构建的口蹄疫-伪狂犬重组病毒TK~-/gG~-/P1~+基因组共转染猪肾细胞IBRS-2,通过空斑纯化得到了新型口蹄疫重组伪狂犬病毒株FHG/P1/PRV。结果表明,此重组病毒能同时表达具有生物学活性的O型口蹄疫衣壳前体蛋白P1以及多抗原合成表位肽FHG,并且伪狂犬病毒基因组上的多点插入表达并不影响其在宿主细胞上的增殖。将此疫苗免疫40日龄的仔猪,结果表明,重组病毒FHG/P1/PRV较之亲本株TK~-/gG~-/P1~+,能够诱导较好的体液免疫和细胞免疫。并且由于FHG/P1/PRV病毒基因组中的gE基因被插入失活,因此该疫苗能够与国际通用的PRV鉴别诊断方法配合使用,这为此疫苗的推广奠定了基础。
2、FMDV DNA疫苗的构建和免疫效果
分别以pcDNA3.1+和pSCA1为载体,构建了表达O型FMDV空衣壳蛋白的常规DNA疫苗表达质粒pceCAP和“自杀性”DNA疫苗表达质粒pSCAI-eCAP,转染BHK-21细胞,Western blot检测证实两种表达质粒均能正确表达FMDV空衣壳蛋白VP0,VP3,和VP1。将pceCAP和pSCAI-eCAP分别以100μg/只免疫Balb/c小鼠,并对这两种疫苗的免疫效果进行了比较,发现“自杀性”DNA疫苗组产生的ELISA抗体和中和抗体明显高于常规DNA疫苗组的,且“自杀性”DNA疫苗能诱导更强的细胞免疫。捕狙⒔峁砻鳌白陨毙浴盌NA疫苗pSCAI-eCAP能完全保护小鼠抵抗FMDV同型强毒的攻击。是一种极具潜力的FMDV候选DNA疫苗。
3、杆状病毒介导的表达FMDV空衣壳蛋白的重组病毒疫苗的构建以及免疫原性的研究
杆状病毒不能感染哺乳动物细胞,但可以进入不同物种和组织来源的多种哺乳动物细胞,并在合适的哺乳动物启动子控制下表达外源基因。目前利用杆状病毒作为体内基因投送的载体已受到广泛的关注。但研究证实血清补体系统是杆状病毒体内基因转导过程中的主要抑制因素。因此,为了消除杆状病毒的补体敏感性,在本研究中利用水泡性口炎病毒G蛋白修饰杆状病毒。将含有CMV启动子驱动的编码FMDV空衣壳蛋白的整个读码框表达的基因克隆到杆状病毒转移载体pFast-VSV-G中,构建重组病毒Ac-V-eCAP。体外转导试验表明,构建的重组病毒均能高效转导哺乳动物细胞.将重组病毒分别以不同的剂量(10~9PFU,10~8PFU,10~7PFU)肌肉注射免疫小鼠,试验结果表明,无论是从体液免疫水平还是细胞免疫水平来比较,重组杆状病毒Ac-V-eCAP以10~9PFU的剂量免疫小鼠时,能够诱导最高的体液免疫水平和细胞免疫水平,具有较好的免疫效果。病毒血症结果表明,Ac-V-eCAP当以10~9PFU的剂量免疫小鼠时能够针对FMDV同型强毒株的攻击提供较好的保护(4/5),但是还是略要低于灭活苗(5/5)。
4、猪源髓细胞分化因子MyD88作为分子佐剂在小鼠体内对FMDV DNA疫苗免疫效果的影响
通过cross-over PCR的方法从猪外周全血中克隆髓细胞分化因子MyD88,并对其序列进行分析。结果获得了髓细胞分化因子MyD88,全长882bp,序列测定及同源性分析表明其与GenBank中报道的牛以及人的髓细胞分化因子基因同源性较高,同源性分别为89%和87%;与人,鼠对应功能域的氨基酸序列的分析结果表明,death区,猪源MyD88与人、鼠源MyD88同源性分别为77.3%,72.7%。TIR区,猪源MyD88与人、鼠源MyD88同源性分别为97.8%,91%。结合分子建模的结果,预测了猪源MyD88蛋白death区位于40-105aa.,TIR区位于158-291aa.。将表达猪源MyD88基因的表达盒插入FMDV DNA疫苗pceCAP中,获得共表达质粒pcMyD88-eCAP,将pcMyD88-eCAP和pceCAP分别以100μg/只免疫Balb/c小鼠,并对这两种疫苗的免疫效果进行了比较,结果无论体液免疫还是细胞免疫,共表达质粒pcMyD88-eCAP较之普通质粒无显著免疫增强效应。这说明猪源MyD88蛋白可能在小鼠体内活性极低,不能激发天然免疫反应。
Foot-and-mouth disease virus(FMDV) is the causative agent of highly infectious and the economically most important animal viral disease affecting cloven hoofed animal in the world.Although mortality associated with FMD is usually low,the disease decreases livestock productivity,and affects countries cannot participate in international trade of animals and animal products.Immunization is the main method to prevent and control this disease.In the present,the available vaccines to prevent FMDV are mainly attenuated and inactivated vaccines.Although attenuated vaccine and inactive are effective to prevent animals from FMDV infection,there exist disadvantages such as,it is expensive and it is dangerous that the virus escapes from vaccine plant.It is urgent to develop more safe and effective vaccine to prevent and control FMD.
Therefore,in the present study,we explored the new vaccine design against FMDV, such as that constructed the recombinant viruses based on pseudorabies virus (PRV)-vector expression system or baculovirus expression system,and suicidal DNA vaccines based on Semiliki Forest virus(SFV)-replicon.Furthermore,the immune efficacy and protection of the candidate vaccines were investigated.Additionally,swine myeloid differentiation factor 88(MyD88) was cloned and studied as FMDV DNA vaccine adjuvants.The most research works were as following:
1.Construction and Immune Response Characterization of a Recombinant
Pseudorabies Virus Co-expressing Capsid Precursor Protein(PI) and a Multiepitope Peptide of Foot-and-mouth Disease Virus in Swine
Foot-and-mouth disease(FMD) is the most contagious and devastating disease of livestock.Our previous studies demonstrated that TK~-/gG~-/P1~+,a recombinant expressing the FMDV capsid precursor protein(P1) based on attenuated pseudorabies virus(PRV) TK~-/gG~-,can be used as a recombinant vaccine to protect pigs against both pseudorabies and FMD.However,because the P1 expression cassette is inserted in the gG locus of the genome of PRV TK~-/gG~-,this bivalent vaccine can not be used in conjunction with the PRV gE-ELISA,an extensively used discriminatory serological test in eradication programes for pseudorabies,which limits the clinical use of this bivalent vaccine.To circumvent this shortcoming,in this study,an expression cassette containing synthetic mulfiepitopes gene "FHG" consisting of six potential B cell epitopes and two potential T cell epitopes of FMDV,under the control of CMV promoter,was further inserted into the gE/gI locus of genome of TK~-/gG~-/P1~+,resulting in a new recombinant FHG/P1/PRV. The immunogenicity of FHG/P1/PRV were evaluated and compared with TK~-/gG~-/P1~+ in piglets.Our results clearly showed that FHG/P1/PRV performed better than or comparable with TK~-/gG~-/P1~+,as demonstrated by comparable PRV-specific neutralizing antibodies,enhanced FMDV-specific neutralizing antibodies and cellular immune responses.More importantly,no gE- and gG-specific antibodies could be detected in pigs immunized with FHG/P1/PRV.These data indicate that FHG/P1/PRV is a promising bivalent vaccine candidate with more extensive potential application than TK~-/gG~-/P1~+ against both pseudorabies and FMD.
2.Construction and immune effect of new DNA vaccines against FMDV
A DNA-based vaccine pceCAP and a suicidal DNA vaccine construct pSCAI-eCAP were generated respectively by subcloning the genes which may express FMDV Virus-Like Particles into expression vector pcDNA3.1+ and expression vector pSCA1.In tranfected BHK-21 cells,pceCAP and pSCAI-eCAP both could express FMDV VLP proteins.After immunization in Balb/c mice,the Pl-specific ELISA antibodies and neutralizing antibodies induced by pSCAI-eCAP was relatively higher than those obtained in mice immunized with the conventional DNA vaccine pceCAP.Further analyses of cell-mediated immune responses showed that pSCAI-eCAP induced higher IFN-γlevel.Collectively these results indicated that suicidal DNA vaccine is an alternative strategy to conventional DNA vaccine to control FMD.
3.Construction and immune effect of recombinant baculovirus expressing the FMDV VLP proteins
Although AcMNPV are failing to replicate in vertebrate cells,it does express aline genes that are dependent on the strength of the promoter used to drive transcription of the foreign gene.Follwing these findings,baculovirus have emerged as a vector with great potential for gene transfer in mammalian cells.However,in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system.Therefore,in this study we describes the generation of a recombinant baculovirus in which the vesicular stomatitis virus glycoprotein G(VSV-G) is present in the viral envelope.This protein was demonstrated to enhance the escape of baculovirus vectors from intracellular endosomes,increasing the transduction efficiency of the virus. The recombinant virus Ac-V-eCAP was constructed to carry the gene which could express FMDV VLPs under the control of the eytomegalovirus immediate-early promoter-enhancer and express vesicular stomatitis virus glycoprotein(VSV-G) in the viral envelope by inserting the VSV-G coding sequence downstream of the polyherdrin promoter.To characterize the induction of antigen-specific immune response mediated by baculovirus,mice were subjected to intramuscular with different doses of baculovirus vectors ranging from 10~8 to 10~(10)pfu/mL in a 100μL volume.Mice immunized with Ac-V-eCAP10~(10)pfu/mL showed the highest humoral immune response lever and cell uar immune response.
What's more,the mice of this group were also protected from challenge(four of five mice were protected).Taken altogether,Ac-V-eCAP with the dose of 10~9pFU shows the best immunogenicity and may be used as a strategy to develop a new genetation of vaccine against FMDV in the future.
4.Cloning swine myeloid differentiation factor 88(MyD88) gene and studying it as FMDV DNA vaccine adjuvants
According to the reported myeloid differentiation factor 88(MyD88) sequence of different species and EST sequence of swine in genbank,pairs of primers were designed to amplify MyD88 gene from swine blood.Firstly,fragments of F1 and F2 which were both containing part of MyD88 and also covering each other partly were amplified by reverse transcription-polymerase chain reaction(RT-PCR).Then using F1 and F2 as template,MyD88 gene was amplified by cross-over PCR and cloned into pMD-18T vector.The product was sequenced and analysed.Nucleotide sequence of MyD88 gene of swine was 882bp in length,encoding 294 amino acids.Gene comparison of the swine MyD88 gene with other known MyD88 gene from different species was performed and analyzed.Corresponding full length of swine MyD88 gene and other reported sequences, it shared the highest identity with Bos taurus and Homo sapiens,the identity was 89%and 87%at the nucleotide level.It was nearly no significantly identity with the MyD88 gene from Branchiostoma belched.It was reported that MyD88 gene had two domains,one was Death-domain,the other was TIR-domain.The molecular modeling toward swine MyD88 also showed the same result.What's more,the amino acid sequence of the two domains was also compared with that of Homo sapiens and Mus musculus,individually. For the Death-domain,the identity of the swine MyD88 gene with the other two species was 77.3%and 72.7%.For the TIR-domaln,the identity of the swine MyD88 gene with the other two species was 97.8%and 91%.Based on all of these results,we may conclude that for the swine MyD88 protein,the Death-domain was at 40-105aa,while the TIR-domain was at 158-291aa.In conclusion,we had cloned the swine MyD88 gene and analysed its sequence both at nucleotide level,also at amino acid lever.We predicted the swine MyD88 protein two domains.All of these work would give us help on study of protein functions.Then we constructed co-expression plasmid pcMyD88-eCAP and immunized it with each mouse at 100μg.The results of immune response test showed that it had rarely stronger response compared to peeCAP.It was might because that MyD88 from swine had low activity in mice and so couldn't induce innate immune response.
鉴于此,本研究对FMDV新型疫苗设计进行了新的探索,以构建了伪狂犬病毒(PRV)为载体的重组PRV-FMDV新型基因工程疫苗,以西门利克森林病毒(SFV)为复制子的“自杀性”DNA疫苗和以杆状病毒为载体的重组病毒疫苗,并对构建的三种候选疫苗进行了免疫效力研究;克隆并表达了猪源髓细胞分化因子MyD88,并探讨了其作为分子佐剂在小鼠体内对FMDV DNA疫苗免疫效果的影响。主要研究内容如下:
1、共表达0型口蹄疫衣壳前体蛋白P1及其多抗原合成表位FHG的重组伪狂犬病毒FHG/P1/PRV的构建和免疫原性研究
根据已公布的O型口蹄疫抗原表位设计合成了多抗原表位肽基因fhg,将其插入到中间转移载体pIECMV中,获得重组中间转移质粒pIEFHG,将pIEFHG与本室构建的口蹄疫-伪狂犬重组病毒TK~-/gG~-/P1~+基因组共转染猪肾细胞IBRS-2,通过空斑纯化得到了新型口蹄疫重组伪狂犬病毒株FHG/P1/PRV。结果表明,此重组病毒能同时表达具有生物学活性的O型口蹄疫衣壳前体蛋白P1以及多抗原合成表位肽FHG,并且伪狂犬病毒基因组上的多点插入表达并不影响其在宿主细胞上的增殖。将此疫苗免疫40日龄的仔猪,结果表明,重组病毒FHG/P1/PRV较之亲本株TK~-/gG~-/P1~+,能够诱导较好的体液免疫和细胞免疫。并且由于FHG/P1/PRV病毒基因组中的gE基因被插入失活,因此该疫苗能够与国际通用的PRV鉴别诊断方法配合使用,这为此疫苗的推广奠定了基础。
2、FMDV DNA疫苗的构建和免疫效果
分别以pcDNA3.1+和pSCA1为载体,构建了表达O型FMDV空衣壳蛋白的常规DNA疫苗表达质粒pceCAP和“自杀性”DNA疫苗表达质粒pSCAI-eCAP,转染BHK-21细胞,Western blot检测证实两种表达质粒均能正确表达FMDV空衣壳蛋白VP0,VP3,和VP1。将pceCAP和pSCAI-eCAP分别以100μg/只免疫Balb/c小鼠,并对这两种疫苗的免疫效果进行了比较,发现“自杀性”DNA疫苗组产生的ELISA抗体和中和抗体明显高于常规DNA疫苗组的,且“自杀性”DNA疫苗能诱导更强的细胞免疫。捕狙⒔峁砻鳌白陨毙浴盌NA疫苗pSCAI-eCAP能完全保护小鼠抵抗FMDV同型强毒的攻击。是一种极具潜力的FMDV候选DNA疫苗。
3、杆状病毒介导的表达FMDV空衣壳蛋白的重组病毒疫苗的构建以及免疫原性的研究
杆状病毒不能感染哺乳动物细胞,但可以进入不同物种和组织来源的多种哺乳动物细胞,并在合适的哺乳动物启动子控制下表达外源基因。目前利用杆状病毒作为体内基因投送的载体已受到广泛的关注。但研究证实血清补体系统是杆状病毒体内基因转导过程中的主要抑制因素。因此,为了消除杆状病毒的补体敏感性,在本研究中利用水泡性口炎病毒G蛋白修饰杆状病毒。将含有CMV启动子驱动的编码FMDV空衣壳蛋白的整个读码框表达的基因克隆到杆状病毒转移载体pFast-VSV-G中,构建重组病毒Ac-V-eCAP。体外转导试验表明,构建的重组病毒均能高效转导哺乳动物细胞.将重组病毒分别以不同的剂量(10~9PFU,10~8PFU,10~7PFU)肌肉注射免疫小鼠,试验结果表明,无论是从体液免疫水平还是细胞免疫水平来比较,重组杆状病毒Ac-V-eCAP以10~9PFU的剂量免疫小鼠时,能够诱导最高的体液免疫水平和细胞免疫水平,具有较好的免疫效果。病毒血症结果表明,Ac-V-eCAP当以10~9PFU的剂量免疫小鼠时能够针对FMDV同型强毒株的攻击提供较好的保护(4/5),但是还是略要低于灭活苗(5/5)。
4、猪源髓细胞分化因子MyD88作为分子佐剂在小鼠体内对FMDV DNA疫苗免疫效果的影响
通过cross-over PCR的方法从猪外周全血中克隆髓细胞分化因子MyD88,并对其序列进行分析。结果获得了髓细胞分化因子MyD88,全长882bp,序列测定及同源性分析表明其与GenBank中报道的牛以及人的髓细胞分化因子基因同源性较高,同源性分别为89%和87%;与人,鼠对应功能域的氨基酸序列的分析结果表明,death区,猪源MyD88与人、鼠源MyD88同源性分别为77.3%,72.7%。TIR区,猪源MyD88与人、鼠源MyD88同源性分别为97.8%,91%。结合分子建模的结果,预测了猪源MyD88蛋白death区位于40-105aa.,TIR区位于158-291aa.。将表达猪源MyD88基因的表达盒插入FMDV DNA疫苗pceCAP中,获得共表达质粒pcMyD88-eCAP,将pcMyD88-eCAP和pceCAP分别以100μg/只免疫Balb/c小鼠,并对这两种疫苗的免疫效果进行了比较,结果无论体液免疫还是细胞免疫,共表达质粒pcMyD88-eCAP较之普通质粒无显著免疫增强效应。这说明猪源MyD88蛋白可能在小鼠体内活性极低,不能激发天然免疫反应。
Foot-and-mouth disease virus(FMDV) is the causative agent of highly infectious and the economically most important animal viral disease affecting cloven hoofed animal in the world.Although mortality associated with FMD is usually low,the disease decreases livestock productivity,and affects countries cannot participate in international trade of animals and animal products.Immunization is the main method to prevent and control this disease.In the present,the available vaccines to prevent FMDV are mainly attenuated and inactivated vaccines.Although attenuated vaccine and inactive are effective to prevent animals from FMDV infection,there exist disadvantages such as,it is expensive and it is dangerous that the virus escapes from vaccine plant.It is urgent to develop more safe and effective vaccine to prevent and control FMD.
Therefore,in the present study,we explored the new vaccine design against FMDV, such as that constructed the recombinant viruses based on pseudorabies virus (PRV)-vector expression system or baculovirus expression system,and suicidal DNA vaccines based on Semiliki Forest virus(SFV)-replicon.Furthermore,the immune efficacy and protection of the candidate vaccines were investigated.Additionally,swine myeloid differentiation factor 88(MyD88) was cloned and studied as FMDV DNA vaccine adjuvants.The most research works were as following:
1.Construction and Immune Response Characterization of a Recombinant
Pseudorabies Virus Co-expressing Capsid Precursor Protein(PI) and a Multiepitope Peptide of Foot-and-mouth Disease Virus in Swine
Foot-and-mouth disease(FMD) is the most contagious and devastating disease of livestock.Our previous studies demonstrated that TK~-/gG~-/P1~+,a recombinant expressing the FMDV capsid precursor protein(P1) based on attenuated pseudorabies virus(PRV) TK~-/gG~-,can be used as a recombinant vaccine to protect pigs against both pseudorabies and FMD.However,because the P1 expression cassette is inserted in the gG locus of the genome of PRV TK~-/gG~-,this bivalent vaccine can not be used in conjunction with the PRV gE-ELISA,an extensively used discriminatory serological test in eradication programes for pseudorabies,which limits the clinical use of this bivalent vaccine.To circumvent this shortcoming,in this study,an expression cassette containing synthetic mulfiepitopes gene "FHG" consisting of six potential B cell epitopes and two potential T cell epitopes of FMDV,under the control of CMV promoter,was further inserted into the gE/gI locus of genome of TK~-/gG~-/P1~+,resulting in a new recombinant FHG/P1/PRV. The immunogenicity of FHG/P1/PRV were evaluated and compared with TK~-/gG~-/P1~+ in piglets.Our results clearly showed that FHG/P1/PRV performed better than or comparable with TK~-/gG~-/P1~+,as demonstrated by comparable PRV-specific neutralizing antibodies,enhanced FMDV-specific neutralizing antibodies and cellular immune responses.More importantly,no gE- and gG-specific antibodies could be detected in pigs immunized with FHG/P1/PRV.These data indicate that FHG/P1/PRV is a promising bivalent vaccine candidate with more extensive potential application than TK~-/gG~-/P1~+ against both pseudorabies and FMD.
2.Construction and immune effect of new DNA vaccines against FMDV
A DNA-based vaccine pceCAP and a suicidal DNA vaccine construct pSCAI-eCAP were generated respectively by subcloning the genes which may express FMDV Virus-Like Particles into expression vector pcDNA3.1+ and expression vector pSCA1.In tranfected BHK-21 cells,pceCAP and pSCAI-eCAP both could express FMDV VLP proteins.After immunization in Balb/c mice,the Pl-specific ELISA antibodies and neutralizing antibodies induced by pSCAI-eCAP was relatively higher than those obtained in mice immunized with the conventional DNA vaccine pceCAP.Further analyses of cell-mediated immune responses showed that pSCAI-eCAP induced higher IFN-γlevel.Collectively these results indicated that suicidal DNA vaccine is an alternative strategy to conventional DNA vaccine to control FMD.
3.Construction and immune effect of recombinant baculovirus expressing the FMDV VLP proteins
Although AcMNPV are failing to replicate in vertebrate cells,it does express aline genes that are dependent on the strength of the promoter used to drive transcription of the foreign gene.Follwing these findings,baculovirus have emerged as a vector with great potential for gene transfer in mammalian cells.However,in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system.Therefore,in this study we describes the generation of a recombinant baculovirus in which the vesicular stomatitis virus glycoprotein G(VSV-G) is present in the viral envelope.This protein was demonstrated to enhance the escape of baculovirus vectors from intracellular endosomes,increasing the transduction efficiency of the virus. The recombinant virus Ac-V-eCAP was constructed to carry the gene which could express FMDV VLPs under the control of the eytomegalovirus immediate-early promoter-enhancer and express vesicular stomatitis virus glycoprotein(VSV-G) in the viral envelope by inserting the VSV-G coding sequence downstream of the polyherdrin promoter.To characterize the induction of antigen-specific immune response mediated by baculovirus,mice were subjected to intramuscular with different doses of baculovirus vectors ranging from 10~8 to 10~(10)pfu/mL in a 100μL volume.Mice immunized with Ac-V-eCAP10~(10)pfu/mL showed the highest humoral immune response lever and cell uar immune response.
What's more,the mice of this group were also protected from challenge(four of five mice were protected).Taken altogether,Ac-V-eCAP with the dose of 10~9pFU shows the best immunogenicity and may be used as a strategy to develop a new genetation of vaccine against FMDV in the future.
4.Cloning swine myeloid differentiation factor 88(MyD88) gene and studying it as FMDV DNA vaccine adjuvants
According to the reported myeloid differentiation factor 88(MyD88) sequence of different species and EST sequence of swine in genbank,pairs of primers were designed to amplify MyD88 gene from swine blood.Firstly,fragments of F1 and F2 which were both containing part of MyD88 and also covering each other partly were amplified by reverse transcription-polymerase chain reaction(RT-PCR).Then using F1 and F2 as template,MyD88 gene was amplified by cross-over PCR and cloned into pMD-18T vector.The product was sequenced and analysed.Nucleotide sequence of MyD88 gene of swine was 882bp in length,encoding 294 amino acids.Gene comparison of the swine MyD88 gene with other known MyD88 gene from different species was performed and analyzed.Corresponding full length of swine MyD88 gene and other reported sequences, it shared the highest identity with Bos taurus and Homo sapiens,the identity was 89%and 87%at the nucleotide level.It was nearly no significantly identity with the MyD88 gene from Branchiostoma belched.It was reported that MyD88 gene had two domains,one was Death-domain,the other was TIR-domain.The molecular modeling toward swine MyD88 also showed the same result.What's more,the amino acid sequence of the two domains was also compared with that of Homo sapiens and Mus musculus,individually. For the Death-domain,the identity of the swine MyD88 gene with the other two species was 77.3%and 72.7%.For the TIR-domaln,the identity of the swine MyD88 gene with the other two species was 97.8%and 91%.Based on all of these results,we may conclude that for the swine MyD88 protein,the Death-domain was at 40-105aa,while the TIR-domain was at 158-291aa.In conclusion,we had cloned the swine MyD88 gene and analysed its sequence both at nucleotide level,also at amino acid lever.We predicted the swine MyD88 protein two domains.All of these work would give us help on study of protein functions.Then we constructed co-expression plasmid pcMyD88-eCAP and immunized it with each mouse at 100μg.The results of immune response test showed that it had rarely stronger response compared to peeCAP.It was might because that MyD88 from swine had low activity in mice and so couldn't induce innate immune response.
引文
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