牛布鲁氏杆菌单克隆抗体的制备及部分特性鉴定

目的 制备牛布鲁氏杆菌单克隆抗体(单抗),并对其部分特性进行鉴定.方法 选用布鲁氏杆菌地方株牛三型菌S85A抗原免疫BALB/c鼠,应用淋巴细胞杂交瘤技术,取脾细胞并与SP2/O骨髓瘤细胞融合,通过间接ELISA筛选,得到能稳定分泌抗牛布鲁氏杆菌单克隆抗体的杂交瘤细胞株,并鉴定其抗体亚类.结果 经3次有限稀释法克隆,间接ELISA筛选,得到4株能稳定分泌抗牛布鲁氏杆菌单克隆抗体的杂交瘤细胞株.抗体亚类分别为3株IgG 2b和1株IgG3.结论 间接ELISA方法证实这些单抗仅与牛种布鲁氏杆菌呈阳性反应,而与其他种的布鲁氏杆菌菌株均无交叉反应.证明得到的细胞株是牛种布鲁氏杆菌单克隆抗体分泌细胞株.     

布鲁氏菌疫苗株S2全基因组测序及牛羊布鲁氏菌比较基因组学研究

目的 为了了解布鲁氏菌S2疫苗株全基因组结构及分子生物学功能。方法 对布鲁氏菌疫苗株S2进行全基因组测序,并与GenBank公布的6株牛羊布鲁氏菌进行比较基因组学研究。结果与结论 通过全基因组测序发现S2基因组大小为3 331 982 bp,预测基因有3 243个,GC含量为57.23 %。S2预测基因中大多数基因功能主要与糖代谢﹑氨基酸代谢﹑氨基酸转运和膜转运有关。通过比较基因组学研究发现S2有20个特有基因。另外S2与GenBank公布的S2序列进行比对发现有19个差异基因。     

多重PCR快速检测鉴别牛布鲁氏菌和牛分枝杆菌的研究与应用

目的利用多重PCR方法建立一种同时快速检测鉴别牛布鲁氏菌和牛分枝杆菌的方法。方法根据牛布鲁氏菌具有种特异性的BCSP-31K基因和牛分枝杆菌23SrRNA，设计并合成了两对分别扩增牛布鲁氏菌和牛分枝杆菌的特异性引物，建立了多重PCR同时快速检测鉴别以上两种病原体的方法。其扩增片段大小牛布鲁氏菌为311bp、牛分枝杆菌为838bp。对所建立的多重PCR方法进行特异性试验和敏感性试验。并应用所建立的方法对临床样品进行检测。结果该多重PCR方法对所有供试的牛布鲁氏菌都能扩增出311bp，结核分枝杆菌都能扩增出838bp目的片段，对其它参试牛的菌株则无311bp和838bp条带，该多重PCR方法对牛布鲁氏菌和牛分枝杆菌的DNA最低检出量为10pg。305份临床样品中10份牛奶样品为牛布鲁氏菌阳性，41份包括36份PPD阳性鼻粘液样品、3份PPD可疑鼻粘液样品和2份PPD阳性牛奶样品为牛分枝杆菌阳性，其余样品均为阴性。     

贵州省2株牛种布鲁氏菌分子流行病学特征分析

目的 分析贵州省牛种布鲁氏菌分子流行病学特征,为牛种布鲁氏菌病疫情的防控提供科学依据。方法 采用BCSP31-PCR及AMOS-PCR技术对经传统方法鉴定为牛种布鲁氏菌的2株分离株进行属/种复核,采用基于布鲁氏菌rpoB基因的单核苷酸多态性分析了解其rpoB基因型,采用MLVA-16技术进行MLVA分型,了解其与国内牛种布鲁氏菌的聚类关系,采用MLST技术分析其序列型及其与国内菌株间的遗传进化关系。结果 2株分离株经BCSP31-PCR和AMOS-PCR技术被鉴定为布鲁氏菌属细菌,未能明确其具体种型;其rpoB基因型相同;MLVA分型与新疆牛种布鲁氏菌共享同一MLVA-16型(4-5-3-12-2-2-3-1-6-43-8-5-5-5-3-3); MLST分析鉴定2株菌均为ST2型,最小间距图显示2株菌与布鲁氏菌属内的牛种布鲁氏菌遗传距离最近,属同一个遗传复合体。结论 贵州省2株牛种布鲁氏菌分离株间的遗传距离近,与新疆牛种3型布鲁氏菌为同一MLVA型,提示分离株引起的感染可能为外省输入。     

分泌牛种布鲁氏菌单克隆抗体杂交瘤细胞A7的悬浮培养研究

牛种布鲁氏菌杂交瘤细胞Ａ７株分泌高中和性的牛种布鲁氏菌单抗ＩｇＧ１，该细菌被培养在Ｔｅｃｈｎｅ－Ｔ１０４ｇａｊｆ１．５升细胞培养装置中，此显示很低的机械剪切力。在悬浮培养时，Ａ７细胞密度可达１．２３×１０６＾６ｍｌ以上，单克隆抗体效价达１Ｌ５１２。     

牛种布鲁氏菌A19-△VirB12标记疫苗双重实时荧光定量PCR方-法的建立

目的 建立一种区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒感染株的双重荧光定量PCR方-法。方法 分别以布鲁氏菌4型分泌系统中VirB8基因、VirB12基因序列设计2对引物及探针,优化实时荧光PCR反应体系及条件。以牛种布鲁氏菌A19-△VirB12标记疫苗株、牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株、大肠杆菌、沙门氏菌基因组DNA进行 Realtime-PCR扩增,评价该方-法特异性。分别构建布鲁氏菌VirB12基因和VirB8基因片段阳性质粒,10倍系列稀释后进行Realtime-PCR扩增,测定该方-法的敏感性。结果 本方-法具有良好的特异性,牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株基因组DNA同时出现VirB8基因与VirB12基因阳性扩增,牛种布鲁氏菌A19-△VirB12标记疫苗株仅出现VirB8基因阳性扩增,大肠杆菌、沙门氏菌均未扩增出目的条带,对VirB8基因及VirB12基因片段阳性质粒的检测限分别为约10² copies/μL和10³ copies/μL。该方-法仅用于鉴别区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒株。结论 本研究建立的布鲁氏菌双重Realtime-PCR方-法,具有良好的特异性和敏感性,为今后鉴别牛种布鲁氏菌A19-△VirB12分子标记疫苗免疫牛与自然感染牛提供技术支撑。     

牛布鲁氏菌抗独特型抗体菌苗对大动物（牛）的抗菌保护研究

用氢氧化铝佐剂配制的牛抗独特型抗体菌苗（简称二抗佐剂苗）免疫大动物牛，通过免疫程序的测定，确定用２种程序，２个剂量进行免疫。免疫后观察其不同时期的血学清免疫反应和保护力。初步结果表明：二抗佐剂苗免疫后与Ｓ１９号苗具有同等的抗原刺激作用，在引发同源抗体的能力上Ｓ１９号苗诱发的血清滴度高于二抗佐剂苗，所做的抗菌保护试验表明，保护力可达６个月以上。     

流产布鲁氏菌omp25基因毕赤酵母表达载体的构建和鉴定

目的构建流产布鲁氏菌omp25基因毕赤酵母分泌型表达载体。方法根据目的基因序列分析结果和毕赤酵母对密码子的优先选择性,选择了抗原性较强的长471bp的基因片段进行克隆和构建流产布鲁氏菌omp25基因毕赤酵母分泌型表达载体,并予鉴定。结果鉴定结果表明目的基因片段与发表的序列完全一致并正确地插入到酵母表达载体pPIC9Kα因子分泌信号肽下游。结论成功地构建了流产布鲁氏菌omp25基因的毕赤酵母表达载体pPIC9K-omp25。     

Immunogencity of HSA-L7/L12 (Brucella abortus Ribosomal Protein) in an Animal Model

Background: The immunogenic Brucella abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. Objective: This study was aimed to evaluate the protection of recombinant Human Serum Albumin (HAS)-L7/L12 fusion protein in Balb/c mice. Methods: The amplified L7/L12 gene was cloned in pYHSA5 vector, pYHSA5-L7/L12 construct was transformed in Saccharomyces cerevisiae and the expressed protein from supernatant was purified by affinity chromatography. Balb/c mice were immunized in five groups by tHSA-L7/L12 fusion protein (group 1), Brucella abortus S19 (group 2), HSA (group 3), recombinant L7/L12 (group 4), PBS (group 5). ELISA to detect antibody production, LTT test to assess antigen specific lymphocyte response were conducted prior to virulent B. abortus strain 544 challenge two weeks after the last injection. Bacterial counts from spleens of immunized mice were done four weeks after challenge. Results: In ELISA tests, the specific antibodies exhibited a dominance of immunoglobulin IgG1 over IgG2a. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference in proliferative response of L7/L12 and tHSA-L7/L12 fusion protein (p>0.05). The L7/L12 and tHSA-L7/L12 fusion protein vaccines could also induce significant protection against challenge with the virulent strain B. abortus 544 in Balb/c mice (p≤0.05). Conclusion: The tHSA-L7/L12 fusion protein, similar to L7/L12 has the ability to induce antigen specific lymphocyte proliferation, stimulate humoral immunity and engender protection.     

活化TLR9的牛种布氏菌基因外重复回文序列筛选及活性检测

目的 筛选具有活化Toll样受体9(TLR9)的牛种布氏菌基因外重复回文序列(Repetitive Extragenic Palindrome Squence, REPs)并检测其活性,为布氏菌病的治疗提供新思路。方法 基于Brucella abortus A13334基因组序列,利用生物信息学技术识别其REPs后,合成序列。将合成的天然骨架的脱氧寡核苷酸(ODNs)转染小鼠单核巨噬细胞株RAW264.7,以ELISA检测IFN-α的分泌水平。采用TLR9-siRNA沉默RAW264.7中的TLR9,将上述诱导IFN-α分泌增加的阳性ODN序列转染RAW264.7,ELISA方法检测IFN-α的分泌变化。结果 筛选出1 857条牛种布氏菌REPs,选择2级茎环结构较好的5条ODNs序列进行合成,ELISA方法检测显示ODNs M4、M5介导IFN-α分泌量显著高于阴性对照(P<0.05),且阳性ODN M5所介导的IFN-α分泌可以被TLR9-siRNA显著抑制。结论 布氏菌基因组中存在可以活化TLR9信号通路的REPs,此结果有助于对布氏菌致病和免疫机制的认识。     

马鹿布氏杆菌25kDa外膜蛋白基因的克隆、序列分析及其表达研究

目的克隆马鹿布氏杆菌ML分离株25kDa外膜蛋白(Omp25)基因,并在大肠杆菌中表达。方法运用RT-PCR技术从马鹿布氏杆菌分离株中扩增出Omp25基因,将其克隆入pMD18-T中进行核苷酸序列测定和遗传变异分析,并将目的基因亚克隆到大肠杆菌表达载体pET28a中诱导表达。结果该基因全长642bp,编码213个氨基酸;其中前23个氨基酸残基构成信号肽。在推导的氨基酸序列中,存在两个跨膜区,但没有潜在的N-联糖基化位点。与GenBank中已经登录的布氏杆菌其他11个分离株相比,马鹿布氏杆菌分离株Omp25基因变异较小,以散在的点突变为主,Omp25基因核苷酸和推导氨基酸序列的同源性分别为99.1%-99.4%和99.2%-99.5%之间。转化重组质粒pETOMP25的大肠杆菌BL21(DE3)在IPTG的诱导下,可表达出具有反应原性的目的蛋白,表达量占菌体蛋白的18.6%。结论马鹿布氏杆菌分离株与流产型布氏杆菌其它分离株Omp25基因同源性很高,可能是一个毒力较强的野毒株。     

布鲁氏菌A19疫苗株全基因组测序及比较基因组学分析

目的 探索布鲁氏杆菌A19疫苗株全基因组的结构、分子生物学的功能,并对其生物信息学进行研究。方法 采用Illumina Hiseq 4000和PacBio对A19进行全基因组测序,并与GenBank 上的8株菌进行比较基因组学解析。A19基因组3 286 167 bp, 预测3 371个基因,GC含量57.25%。通过注释COG库,对应基因有2 560个,将其归入22类COG中;根据比对KEGG库,得到2 544个基因,共参与33类代谢通路。结果 综合两个数据库结果发现,大多数A19预测基因中的基因功能主要与膜运输、氨基酸转运及碳水化合物代谢有关。结论 通过分析发现, A19和猪羊牛种布鲁氏菌之间存在一定差异,并找出牛种毒力基因。本实验通过测序A19全基因组,为布鲁菌疫苗的研究提供思路。     

羊布鲁杆菌脂蛋白OMP19单克隆抗体制备与鉴定

目的 制备与鉴定羊布鲁杆菌脂蛋白OMP19单克隆抗体,用于布菌感染免疫机制研究。方法 将OMP19基因连接入pET-30a(+)表达载体中,构建pET-30a(+)/OMP19质粒,转化入大肠埃希氏菌BL21(E3),以不同浓度异丙基-β-D硫代半乳糖苷(IPTG)进行诱导表达,采用镍金属螯合亲和层析(NI-NTA)纯化;以布菌阳性血清检测重组蛋白免疫反应性,并利用杂交瘤技术制备单克隆抗体,以天然OMP19蛋白及布菌外膜蛋白提取物(NMP)对制备的单克隆抗体进行Western Blot及酶免疫染色试验(IEST)鉴定。结果 表达了OMP19蛋白,分子量约19 kDa,通过纯化纯度可达95%,Western Blot分析显示蛋白具有良好的抗原性,并制备了OMP19抗原23株鼠源性单克隆抗体,鉴定结果 显示22(95.65%)株能与天然OMP19蛋白反应,18(78.26%)株能与NMP反应,其中IgG1(k)亚型占91.30%;4株能与羊种布鲁氏菌菌涂片反应。结论 成功制备具有良好抗原性的重组OMP19蛋白,筛选出识别天然蛋白的单克隆抗体,已初步应用于布菌的检测,为OMP19抗原B细胞表位的筛选奠定基础。     

布鲁氏菌组分抗原E化学实质的研究

从羊种布鲁氏菌16M中提出的化学组分抗原E包含了菌细胞中大分子蛋白、核酸、完整的菌壁多糖及脂多糖，通过化学处理及修饰提取方法可不同程度使脂多糖丢失。结合动物试验结果，揭示出脂多糖与E抗原免疫原性的内在关系。     

布鲁杆菌基因工程疫苗免疫保护效率的初步观察

目的比较布鲁杆菌6种抗原表位所获得的基因工程疫苗的免疫保护效率。方法布鲁杆菌核糖体蛋白L7/L12、胞质蛋白P39、细胞表面蛋白BCSP31、二氧四氢喋啶合成酶BLS、16.5×103的外膜蛋白PAL和翻译起始因子IF3的基因片段与真核表达载体pcDNA3.1( )构建的核酸疫苗及上述6条片段转入pET32a( )后诱导表达的的重组蛋白免疫小鼠,3次免疫后进行攻毒实验,对其免疫保护效率进行初步观察。结果L7/L12和BLS的重组蛋白疫苗和核酸疫苗都产生了非常显著的保护作用,P39的核酸疫苗、IF3和BCSP31的重组蛋白疫苗也产生了非常显著的保护作用,其中L7/L12核酸疫苗的保护效率最高。结论初步认为布鲁杆菌的优势抗原表位所获得的基因工程疫苗可产生一定的抗布鲁杆菌作用,可作为未来布鲁杆菌新型疫苗的候选者,有进一步研究的价值。     

Maintenance of ribosomal protein S19 in plasma by complex formation with prothrombin

We have demonstrated that the cross-linking of ribosomal protein S19 (RP S19) on platelets by activated factor XIII provides chemotactic potency to monocytes/macrophages for a resolution of coagulum. Factor XIII is activated by an active form of prothrombin, thrombin. We here report that RP S19 is present as a complex with prothrombin in the blood stream. Formation of this complex was blocked by a mutation of the glycosaminoglycan-binding basic cluster (Lys(23) -Lys(29) ) in RP S19. Prothrombin-RP S19 interaction was enhanced by an absence of Ca(2+) and the plasma RP S19 concentration was significantly low in the patient treated with warfarin, indicating participation of the γ-carboxyl glutamic acid domain of prothrombin making a salt bridge with the basic cluster. The complex formation likely explains why a protein as small as RP S19 can prevent from a filtering system of renal glomeruli at a steady state. The translocation of RP S19 from prothrombin to platelets during blood coagulation seems to be also advantageous for RP S19 from the perspective of oligomerisation by activated factor XIII, which should have been activated by thrombin.     

Dysregulation of Protein S in COVID-19

Coronavirus Disease 2019 (COVID-19) has been widely associated with increased thrombotic risk, with many different proposed mechanisms. One such mechanism is acquired deficiency of protein S (PS), a plasma protein that regulates coagulation and inflammatory processes, including complement activation and efferocytosis. Acquired PS deficiency is common in patients with severe viral infections and has been reported in multiple studies of COVID-19. This deficiency may be caused by consumption, degradation, or clearance of the protein, by decreased synthesis, or by binding of PS to other plasma proteins, which block its anticoagulant activity. Here, we review the functions of PS, the evidence of acquired PS deficiency in COVID-19 patients, the potential mechanisms of PS deficiency, and the evidence that those mechanisms may be occurring in COVID-19.     

Immunogenicity Following Administration of BNT162b2 and Ad26.COV2.S COVID-19 Vaccines in the Pregnant Population during the Third Trimester

Globally, COVID-19 vaccines are currently being used to prevent transmission and to reduce morbidity and death associated with SARS-CoV-2 infection. Current research reveals that vaccines such as BNT162b2 and Ad26.COV2.S are highly immunogenic and have high short-term effectiveness for most of the known viral variants. Clinical trials showed satisfying results in the general population, but the reluctance in testing and vaccinating pregnant women left this category with little evidence regarding the safety, efficacy, and immunogenicity following COVID-19 vaccination. With the worldwide incidence of COVID-19 remaining high and the possibility of new transmissible SARS-CoV-2 mutations, data on vaccination effectiveness and antibody dynamics in pregnant patients are critical for determining the need for special care or further booster doses. An observational study was developed to evaluate pregnant women receiving the complete COVID-19 vaccination scheme using the BNT162b2 and Ad26.COV2.S, and determine pregnancy-related outcomes in the mothers and their newborns, as well as determining adverse events after vaccination and immunogenicity of vaccines during four months. There were no abnormal findings in pregnancy and newborn characteristics comparing vaccinated versus unvaccinated pregnant women. COVID-19 seropositive pregnant women had significantly higher spike antibody titers than seronegative patients with similar characteristics, although they were more likely to develop fever and lymphadenopathy following vaccination. The same group of pregnant women showed no statistically significant differences in antibody titers during a 4-month period when compared with case-matched non-pregnant women. The BNT162b2 and Ad26.COV2.S vaccines are safe to administer during the third trimester of pregnancy, while their safety, efficacy, and immunogenicity remain similar to those of the general population.     

Identification of a new in-frame deletion of six amino acids in ribosomal protein S19 in a patient with Diamond-Blackfan anemia

Ribosomal protein S19 (RPS19) is currently the only gene associated with Diamond-Blackfan anemia (DBA), a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. RPS19 is mutated in 25% of DBA patients, but its role in DBA pathogenesis remains elusive. We have identified a novel heterozygous microdeletion in RPS19 in a DBA patient presenting with profound anemia after birth. The deletion of 18 nucleotides (233-250; A in start codon is +1) in exon 4 leads to the elimination of 6 amino acids 78IYGGRQ83, affecting the most conserved stretch of three amino acids (YGG) in RPS19. The mutated allele was not detected in the patient's family members, indicating de novo mutation. Both alleles were expressed at the same level. Using an immunofluorescence technique, the mutated RPS19 protein localized to nucleoli, and its intracellular distribution did not differ from the wild-type RPS19. The deletion of only a few amino acids of this protein with a preserved reading frame is rare, and this type of a mutation could be very helpful in further experiments to define the role of the RPS19 protein in DBA pathogenesis.     

Familial transient erythroblastopenia of childhood is associated with the chromosome 19q13.2 region but not caused by mutations in coding sequences of the ribosomal protein S19 (RPS19) gene

Transient erythroblastopenia of childhood (TEC) is a rare condition, which at onset may be difficult to distinguish from Diamond-Blackfan anaemia (DBA). We have previously shown that mutations in the ribosomal protein S19 gene (RPS19) cause DBA. In order to clarify whether TEC and DBA are allelic, we investigated the segregation of markers spanning the RPS19 gene region on chromosome 19q13.2 and performed sequence analysis of all exons in the RPS19 gene in seven TEC sibling pairs. Linkage analysis supported allelism for TEC and DBA at the RPS19 gene locus and implies molecular mechanisms other than structural mutations in the RPS19 gene.