牛支原体新疆分离株P48基因的克隆、原核表达及重组蛋白免疫原性研究

试验旨在确定牛支原体P48基因的免疫原性，为进一步筛选牛支原体免疫保护性基因奠定基础。本研究以牛支原体新疆分离株为研究对象，运用Overlap PCR方法扩增得到点突变后的牛支原体新疆分离株P48基因，构建原核表达载体pET-32a （+）-P48，转化大肠杆菌BL21（DE3）感受态细胞，在诱导剂ITPG的诱导下获得重组蛋白P48，纯化后的重组P48蛋白免疫BALB/c小鼠制备多克隆抗体，运用Western blotting和ELISA方法验证其反应原性和免疫原性。结果表明，试验成功构建原核表达载体pET-32a （+）-P48，重组蛋白P48大小约为66 ku，纯化后的牛支原体P48重组蛋白免疫小鼠后可产生良好的免疫反应，血清抗体滴度达到较高水平（D_{450 nm}值为1.126）。Western blotting结果显示，抗牛支原体P48重组蛋白的鼠血清与牛支原体P48重组蛋白及牛支原体全菌蛋白抗原均能产生明显的抗原抗体反应，表明P48重组蛋白具有良好的免疫原性与反应原性，可作为牛支原体新型疫苗的候选基因，且牛支原体新疆分离株P48基因与国内外5株牛支原体P48基因的同源性很高，亲缘关系较近。     

罗非鱼无乳链球菌LrrG-Sip融合蛋白免疫原性研究

为探究无乳链球菌LrrG(Leucine-rich repeat protein from GBS)和表面免疫原性蛋白Sip(surface immunogenic protein)串联表达的LrrG-Sip重组融合蛋白的免疫原性,该研究将原核表达的LrrG-Sip重组融合蛋白分别以0.5μg·g~(-1)(R1组)、1.0μg·g~(-1)(R2组)和1.5μg·g-1(R3组)每尾200μL腹腔注射免疫尼罗罗非鱼(Oreochromis niloticus),同时以Sip蛋白(S组)、LrrG蛋白(L组)以及PBS(P组)作为对照。2周后对所有免疫鱼体进行无乳链球菌(Streptococcus agalactiae)人工攻毒,攻毒剂量为其半致死浓度(LD_(50):4.0×108CFU·mL~(-1))。结果显示LrrG-Sip重组融合蛋白R1组对尼罗罗非鱼的相对免疫保护率最高,达89.14%;且免疫后第14和第28天,该组鱼体血清抗体OD_(450nm)值分别达0.63和0.64,均显著高于单一蛋白对照组(S和L组)和PBS组(P0.05);R1组鱼体血清过氧化物酶(POD)和碱性磷酸酶(AKP)活性在上述2个时间点也显著高于其他组(P0.05);但溶菌酶(LZM)和总超氧化物歧化酶(T-SOD)活性与其他组之间差异不显著(P0.05)。初步表明LrrG-Sip重组融合蛋白具有良好的免疫原性,其免疫原性明显优于单个蛋白,且能有效减少免疫剂量。     

鮰爱德华菌外膜微孔蛋白ompN1、ompN2和ompN3基因生物信息学分析及截段基因的原核表达与其抗原性检测

根据GenBank中鮰爱德华菌(Edwardsiella ictaluri)外膜微孔蛋白N(outer membrane porin protein,OMP)基因序列(NC_012779.2)设计引物,以鮰爱德华菌Ms12基因组DNA为模板,经PCR扩增后克隆至pMD19-T载体,将阳性克隆菌送公司测序。测序结果通过生物信息学软件对ompN1、ompN2和ompN3序列的氨基酸序列相对分子质量、分子式、信号肽、跨膜区、亲/疏水性、B细胞表位进行预测,并构建系统发育树;根据去信号肽的B细胞表位相对集中区域设计特异性引物,经PCR扩增后,将截段序列克隆至pMD19-T载体,鉴定成功后克隆至原核表达载体pET32a(+)并转化至BL21(DE3)/Rosseta(DE3)进行IPTG诱导表达,最后用兔抗鮰爱德华菌血清对重组蛋白OmpN1、OmpN2和OmpN3进行免疫原性检测。结果显示扩增得到ompN1(1 143bp)、ompN2(1 056bp)和ompN3(1 053bp)的序列,获得的基因登录号分别为KJ831564、KJ831565和KJ831566。经生物信息学分析显示,OmpN1、OmpN2和OmpN3蛋白分子式分别为C1940H2993N543O622S7、C1817H2723N497O579S5和C1896H2843N519O563S7,相对分子质量大小分别为44 100、40 900和41 800,均为含有1个信号肽和跨膜区的疏水性蛋白,具有1个革兰阴性细菌OM_channels超家族结构域,是1种嵌入型的膜外蛋白;SDS-PAGE电泳检测发现,诱导表达的融合重组蛋白均以包涵体的形式出现在沉淀中,大小分别约为61 800、58 700和59 100;Western-blot分析表明,重组蛋白OmpN1、OmpN2和OmpN3均能与兔抗鮰爱德华菌全菌血清结合,表明鮰爱德华菌外膜微孔蛋白N具有亚单位疫苗开发的潜力。     

Isolation and Identification of one Strain of H9 Subtype Avian Influenza Virus and Study on its Biological Characteristics

In 2013,one case of suspected H9 subtype avian influenza occurred in a chicken farm of Jilin povince.Clinical samples were collected from the diseased farm,inoculated into the allantoic cavity of 9-day-old SPF chicken embryo,and then one strain of virus was isolated.The results of HA test,HI test and molecular biology test all showed that the isolate belonged to H9 subtype avian influenza virus (AIV).The HA cleavage site of the isolate was RSSR↓GLF,which was consisted with the molecular characteristic of low pathogenic AIV.The HA peptide chain had 9 potential glycosylation sites which were same as other isolates of recent years.The isolate had 8 receptor binding sites,including 234 receptor binding site by glutamine (Q) mutating into threonine (T).The phylogenetic tree revealed the isolate belonged to Eurasian lineages and it had far genetic relationship with the earliest domestic isolate (A/Chicken/Beijing/1/94(H9N2)),but had close genetic relationship with the representative strain (A/Chicken/Guangxi/55/2005(H9N2)) of major epidemic branch since 2007.We prepared an inactivated oil-emulsion vaccine of the isolate,and then vaccinated SPF chicken.21 days after vaccination,the HI titer of chicken serum antibody reached up to 10log2.The result suggested the isolate had good immunogenicity.     

鸡传染性贫血病毒VP1、VP2基因的表达及免疫原性研究

为探索鸡传染性贫血重组蛋白亚单位疫苗的可行性,将鸡传染性贫血病毒基因分别克隆到转移载体p Fast Bac HTA中,再将其分别转化DH10Bac感受态细胞得到相应的重组表达载体,转染昆虫细胞Sf21获得含有VP1、VP2基因的重组杆状病毒v Bac-VP1、v Bac-VP2,应用悬浮培养的Sf21细胞表达重组蛋白。SDS-PAGE及Western blotting结果证明,VP1、VP2基因在昆虫细胞中得到了表达,动物试验进一步证实,重组蛋白免疫SPF鸡可产生ELISA抗体,提示杆状病毒表达的VP1、VP2具有良好的免疫原性。     

表达牛病毒性腹泻病毒E2蛋白的重组乳酸菌的构建及蛋白免疫原性分析

试验旨在构建能表达牛病毒性腹泻病毒（Bovine viral diarrhea virus，BVDV）E2抗原蛋白的重组乳酸乳球菌（Lactococcus lactis），为进一步研制BVDV乳酸菌口服活载体疫苗奠定基础。将BVDV E2基因克隆后测序，根据乳酸乳球菌的密码子偏嗜性进行优化，再将优化的基因片段插入表达载体pNZ8148中，并电转化乳酸乳球菌NZ9000感受态细胞，构建重组乳酸菌pNZ8148-E2/NZ9000，经1 ng/mL乳链菌肽诱导表达后，对菌体物进行了SDS-PAGE和Western blotting分析。将重组乳酸菌pNZ8148-E2/NZ9000口服免疫6~12月龄健康犊牛，在免疫后不同时间点采集血液样品并分离血清，用间接ELISA方法检测抗体水平。结果显示，PCR扩增到了1 149 bp的目的片段，乳酸菌密码子偏嗜性优化后，GC含量从45.28%变为34.30%。重组质粒pNZ8148-E2经酶切鉴定插入片段与预期大小相符，在菌体裂解物中出现大小约42 ku的条带，与预期蛋白大小一致，且该蛋白可与BVDV E2抗体反应。在免疫犊牛的血清中检测到特异性抗BVDV E2蛋白的抗体。本研究结果表明，表达BVDV E2蛋白的重组乳酸菌口服免疫可诱导犊牛产生特异性的体液免疫反应，该重组菌具有较好的免疫原性。     

巨型艾美耳球虫偏菱形样蛋白EmRP免疫原性分析

在前期研究中,从巨型艾美耳球虫(Eimeria maxima)cDNA文库中筛选到了免疫保护性抗原偏菱形样蛋白EmRP,本研究为揭示该抗原的免疫保护机理,进一步检测了其免疫原性。以E.maxima卵囊cDNA为模板,用RT-PCR技术扩增EmRP,并构建真核表达质粒pVAX1-EmRP,将其经腿部肌肉注射2周龄的雏鸡,3周龄加强免疫。分别于首次免疫后和加强免疫后1周,采集血清,用ELISA方法检测血清特异性IgG水平,用荧光定量PCR方法(qPCR)检测细胞因子水平,用流式细胞术检测CD4^+T淋巴细胞和CD8^+T淋巴细胞的比例,分析EmRP诱导的免疫反应。序列分析表明,EmRP含有偏菱形样蛋白超家族成员保守区域,为非跨膜蛋白,分子量约为28.4 kD。真核表达质粒pVAX1-EmRP免疫鸡后,与对照组相比,鸡体IFN-γ、IL-2、IL-10和IL-17等细胞因子转录水平显著提升;CD8^+和CD4^+ T细胞比例显著提高,而血清特异性IgG水平与对照组差异不显著。结果表明,EmRP诱导的免疫保护作用主要是由其诱导的细胞免疫反应实现的,可以作为研制E.maxima新型疫苗的候选抗原。     

Study on Secretory Expression and Immunogenicity of PPRV F Gene in Insect Cells

This study was aimed to explore the use of baculovirus expression system to secrete peste des petits ruminants virus (PPRV) F gene protein and use it as subunit vaccine. The gene fragment encoding F protein of PPRV was cloned into the baculovirus pFastBac Ⅰ transfer vector with a honeybee melittin signal peptide.The constructed F-pFastBac was transformed into Escherichia coli DH10Bac,resulting the recombinant baculovirus DNA (F-Bacmid) which was confirmed by blue-white plaque assay and antibiotic resistance selection.The F-Bacmid was then transfected into Sf9 insect cells by the cellfectin transfection reagent.The recombinant F protein was expressed in High Five cells in the serum-free medium.The SDS-PAGE and Western blotting analysis of recombinant protein showed that the protein could be expressed in insect cells and secreted into the culture medium. For the immunogenicity study,the recombinant protein was then inoculated into BALB/c mice, the results showed that the recombinant protein was able to stimulate B cells to produce special antibodies. In conclusions,the recombinant baculovirus expressing F protein of PPRV were successfully constructed.This study applied a basis for the development of PPRV subunit vaccine.     

Study on Expression and Preliminary Immunocompetence of CIC Protein Containing Molecular Adjuvants

To express CTB-IsdBid-Clfais(CIC) protein and evaluate its immunogenicity, CTB (as a molecular adjuvant) could be tandem linked with IsdBid-Clfais gene by the overlapping PCR method, then CTB-IsdBid-Clfais(CIC) was inserted into pET-32a(+) vector to construct recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais. The recombinant plasmids pET-32a(+)-CTB-IsdBid-Clfais were transformed into Escherichia coli (E.coli) BL21 to express the CTB-IsdBid-Clfa(CIC) protein, the CIC expression protein and its immune activity were detected by Western blotting and ELISA,respectively. The results showed that the length of CIC gene were 2 072 bp, and CIC was correctly inserted into the pET-32a(+) plasmids, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed. Western blotting result confirmed that the molecular weight of CIC proteins was 95.9 ku, which were correctly expressed by E.coli BL21 with pET-32a (+)-CTB-IsdBid-Clfais plasmids. ELISA results showed that there was no significant difference among the CIC, IsdBid and Clfais protein groups (P>0.05), and there was extremely significant difference between CIC and BSA protein groups (P<0.01). In conclusion, the pET-32a(+)-CTB-IsdBid-Clfais recombinant plasmids were successfully constructed, CIC proteins were correctly expressed, and were able to react with serum from mice immunized with IsdBid and Clfais,respectively,therefore, CIC proteins had strong immune activity.     

鲁氏耶尔森氏菌外膜蛋白ompF基因的分子克隆、生物信息学与免疫原性分析

为筛选潜在的保护性抗原基因研制鲁氏耶尔森氏菌(Yersinia ruckeri)基因工程疫苗,实验利用特异性引物扩增分离自斑点叉尾(Ictalurus punctatus)的Y.ruckeri外膜蛋白omp F基因并对其进行分子克隆,应用相关软件和程序对其核苷酸及氨基酸序列进行生物信息学分析,并进行了omp F蛋白的表达和免疫原性检测。结果显示,omp F基因全长1 095 bp(Gen Bank登录号KP159420),包含一个编码364个氨基酸的完整开放阅读框,其氨基酸序列具有极高保守性,与Y.ruckeri外膜穿孔蛋白omp F(Gen Bank登录号ADK27779.1)亲缘关系最近,序列一致性为99.2%,进化树聚为一支;具有1个革兰氏阴性菌穿孔蛋白保守结构域,存在1个信号肽和1个跨膜区,是一种跨膜蛋白;具有12个与免疫相关的抗原决定簇区域。SDS-PAGE分析发现该蛋白主要以包涵体形式表达在沉淀,经Western-bolt显示omp F蛋白具有较好的免疫原性。以上结果表明omp F基因可作为Y.ruckeri基因工程疫苗的候选抗原基因。