奶牛乳腺炎无乳链球菌临床分离株fbsA基因的克隆与序列分析

目的克隆并分析牛乳腺炎无乳链球菌内蒙古地区临床分离株表面蛋白fbsA基因核苷酸及编码氨基酸序列,预测其潜在的抗原表位。方法以无乳链球菌内蒙古地区临床分离株为材料,根据GenBank中公布的无乳链球菌fbsA基因序列设计特异性克隆引物,采用同源克隆法,PCR扩增fbsA基因序列,采用DNA Star生物信息学软件预测分析其编码氨基酸的潜在抗原性。结果克隆的fbsA基因序列大小为321bp,编码107个氨基酸残基,与GenBank中公布的B群无乳链球菌fbsA基因核苷酸序列同源性为98.13%,氨基酸序列同源性为99%。预测克隆基因编码氨基酸抗原性指数良好。结论成功克隆出内蒙古地区奶牛乳腺炎无乳链球菌表面蛋白fbsA基因序列,并预测其编码氨基酸具有潜在抗原性为进一步研究其原核表达产物的抗原性及致病机制奠定了基础。     

无乳链球菌流行现状与耐药性研究进展

无乳链球菌是一种条件致病菌,可以通过多种途径进行传播。它是引起孕妇、新生儿和老年人等高危群体发病和死亡的重要病原菌,可导致严重的临床感染,在成人中引起的侵袭性感染性疾病发病率有持续上升的趋势。目前无乳链球菌已鉴定出多种序列型、克隆复合物以及10种血清型,它们在不同人群和不同地区的流行状况不尽相同。另外,近年无乳链球菌对抗生素耐药形势愈发严峻,不仅对红霉素、克林霉素等二线抗生素耐药率长期处于高位,甚至对青霉素耐药菌株的报道都已屡见不鲜,这使得无乳链球菌感染的临床治疗面临更多挑战。本文主要对无乳链球菌的流行现状以及对抗生素耐药性研究进展进行综述,以期更加清晰地认识无乳链球菌并为其科学防控提供参考依据。     

牛乳腺炎金黄葡萄球菌肠毒素B基因的克隆及序列分析

目的克隆牛乳腺炎金黄葡萄球菌B(SEB)基因,并进行序列分析。方法根据GenBank公布的金黄葡萄球菌基因的全序列,利用生物学软件Primer5.0和Oligo 6.0设计1对特异性引物,扩增临床分离菌株的SEB基因片段。结果扩增的基因片序列长度为466bp,与标准菌株(CMCC26074)的SEB基因片段序列相似性为100%,与GenBank公布的金黄葡萄球菌菌株(AB479118.1)SEB基因序列相似性为99.93%。结论成功克隆出牛乳腺炎金黄葡萄球菌SEB基因,为建立相应的PCR快速检测牛乳SEB基因方法奠定了基础。     

牛乳腺炎无乳链球菌PI-2a菌毛岛屿BP亚基单克隆抗体胶体金标记试纸条制备

目的 本研究通过优化牛乳腺炎无乳链球菌PI-2a菌毛岛屿BP亚单位单克隆抗体的性能,制备胶体金标记检测牛乳腺炎无乳链球菌的试纸条。方法 利用pET30a(+)-BP重组质粒表达蛋白纯化物免疫小鼠,无菌取脾细胞与sp2/0骨髓瘤细胞融合,筛选杂交瘤细胞,制备单克隆抗体,对单克隆抗体进行胶体金标记后制备检测试剂盒,并确定其灵敏度、特异性等技术指标。结果 经数轮筛选,获得1株阳性杂交瘤细胞株,抗体效价达到1∶256 00。胶体金标记试纸条灵敏度量达到10⁶ CFU/mL。特异性测试结果表明,胶体金标记试纸条与金黄色葡萄球菌、大肠杆菌、沙门氏菌、副结核杆菌、化脓性链球菌培养物人工污染的牛乳样均无阳性反应,只与无乳链球菌乳样出现阳性结果。结论 该胶体金标记免疫层析试纸条,具有实际应用价值,将为牛乳腺炎的筛查检测提供新型、便捷的技术产品。     

无乳链球菌荚膜多糖疫苗的研究进展

无乳链球菌(Streptococcus agalactiae)是一种分布广泛的人兽共患性致病菌。新生儿感染可引起致命性的败血症和脑膜炎。母体免疫是预防新生儿无乳链球菌感染的最有前景的有效策略。然而,目前尚无可用的疫苗。作为无乳链球菌最重要的毒力因子之一,荚膜多糖(CPS)被认为是开发无乳链球菌疫苗的主要靶标。在此,我们综述了无乳链球菌CPS疫苗和CPS-蛋白结合疫苗的研究概况以及最新进展,旨在为无乳链球菌疫苗的研究提供参考。     

弓形虫胚层发育相关蛋白基因的克隆和序列分析

目的 对15个弓形虫虫株的弓形虫胚层发育相关蛋白(TgERP)基因进行克隆和序列分析,了解其变异和进化情况,为评价该蛋白在血清学诊断和疫苗防疫应用上的价值提供理论基础。方法 设计TgERP基因的PCR引物,对15个弓形虫虫株进行PCR扩增和序列测定,测序结果利用Puzzle 5.2、Paup 4.0和DNAStar 5.0软件进行分析。结果 TgERP基因包含的完整开放阅读框(ORF)长度均为315 bp,A+T含量在46.67%～46.98%之间,仅有1个碱基发生变异,变异率为0%～0.32%。编码104个氨基酸,变异率在0%～0.96%之间。系统进化分析显示,TgERP基因序列虽然能够将弓形虫I型虫株与II/III型虫株区分开,但却不能区分所有基因型的虫株。结论 TgERP基因在各基因型虫株中十分保守,不适合作为遗传标记分子来区分不同基因型的弓形虫虫株。     

牛乳腺炎无乳链球菌β溶血素CylE基因对牛巨噬细胞炎性细胞因子的转录和表达及对信号通路MAPK的影响机制研究北大核心CSCD

目的探讨牛乳腺炎无乳链球菌β溶血素CylE基因对牛巨噬细胞吞噬功能的免疫调理分子机制。方法利用牛乳腺炎无乳链球菌临床分离野生菌株及β溶血素CylE基因缺失突变株对牛巨噬细胞进行侵染,提取侵染细胞的总mRNA;利用qPCR检测细胞因子Il-8、Il-10、Il-12、TNFα及NOS2的转录水平,ELISA检测相应表达水平。在此基础上,利用免疫印迹实验鉴定无乳链球菌β溶血CylE基因对MAPK信号通路下游接头分子p-38、p44/42的表达及其磷酸化水平的影响,以明确其调控该信号通路的分子机制。结果巨噬细胞-GBS^(WI)组与巨噬细胞-GBS^(ΔcyIE)组相比,IL-10、TNF-α细胞因子上调,而IL-8、IL-12、NOS2细胞因子表达水平降低,表明无乳链球菌β溶血素CylE对细胞因子IL-10、TNF-α的影响结果一致,相对转录水平均上调,同时抑制细胞因子IL-8、IL-12及NOS2的转录。巨噬细胞-GBS;组与巨噬细胞-GBS;组相比,IL-10及TNF-α的相对表达量增加,而IL-8及IL-12的相对表达量下调,差异均具有统计学意义(均P>0.05),NOS2的相对表达量降低。对MAPK信号通路下游分子磷酸化检测结果显示,无乳链球菌β溶血素CylE基因可激活其下游接头分子p38磷酸化为p-p38,不能激活下游分子p44/42。结论根据上述结果,p38 MAPK是无乳链球菌溶血素CylE基因发挥免疫调控的识别途径,可能成为对其进行识别钝化,实现信号通路封闭,提高对牛乳腺炎预防与治疗有效性的新靶位,为有效防控该病原菌的危害,保障乳源安全提供新的思路和有效途径。     

苏云金杆菌cryIVD基因的克隆及序列分析

目的 克隆苏云金杆菌（B.t.i)cryIVD基因并测定其序列。方法 根据B.t.i cryIVD已知序列，设计合成了1对引物，应用PCR技术扩增cryIVD基因，克隆入pUC18载体，阳性克隆的重组质粒经酶切，电泳鉴定后进行序列测定。结果 PCR扩增得到特异的2.0kb基因片段。重组质粒经酶切后得到预期大小的基因片段。序列分析证明目的基因被完整且正向克隆。结论 B.t.icryIVD基因被成功克隆。     

H9亚型禽流感病毒HA基因的克隆与序列分析

目的扩增、克隆H9N2 HA基因,并进行序列分析,为H9N2流感病毒种间及跨种传播机制研究奠定基础。方法设计特异性引物,扩增克隆H9N2亚型禽流感病毒,测序后进行序列分析。结果成功克隆出4株H9N2亚型禽流感病毒,全长均为1.7 kb,ORF为1 683 bp,编码560个氨基酸;联机检索,与参考毒株比对核苷酸和氨基酸同源性均在90%以上。结论成功克隆4株H9N2亚型禽流感病毒,序列分析显示其核苷酸和推导氨基酸与参考毒株高度同源。     

中华按蚊抗菌肽defensinA编码基因cDNA克隆与序列分析

目的克隆中华按蚊defensin编码基因，并对其序列进行分析。方法提取中华按蚊总RNA。据文献报道合成1对引物deft、def2，RTPCR扩增中华按蚊defensin编码基因，克隆于T-载体，序列测定与分析。结果RT—PCR扩增中华按蚊cDNA获大约308bp片段，测定目的片段序列，结果显示，克隆基因cDNA序列长度为308bp，推导编码64个氨基酸，序列分析显示中华按蚊defensin编码基因与冈比亚按蚊defensin编码基因有高度同源性（95％）。分析结果显示，中华按蚊defensin编码基因为新基因，定名为中华按蚊defensinA。结论克隆出中华按蚊defensinA编码基因，为进一步研究该基因打下了基础。     

Infectious native valve endocarditis by Streptococcus agalactiae species: Case report of pathogen identification only through metagenomic sequencing technology

Introduction:Streptococcus agalactiae is a common pathogen in infective endocarditis, but the positive rate of traditional blood culture diagnosis is not high. It is challenging to obtain a good outcome in the absence of pathogen information for patients with infectious endocarditis.Patient concerns and diagnosis:Here, we report the case of a patient with infective endocarditis caused by S. agalactiae. The initial manifestations of this patient were coma, urinary incontinence, and fecal incontinence and had no history of heart disease or infectious diseases before admission.Interventions and outcomes:When the blood culture was negative 3 consecutive times, the pathogen S. agalactiae was diagnosed in a timely and accurate manner by metagenome sequencing. Eventually, the patient was discharged following surgery and antibiotic treatment.Conclusions:For IE patients with infective endocarditis, metagenome sequencing is a valuable and selective tool for rapid, sensitive, and accurate pathogen detection, especially when the blood culture is negative.     

Characterization of Clinical and Carrier Streptococcus agalactiae and Prophage Contribution to the Strain Variability

Streptococcus agalactiae (group B Streptococcus, GBS) represents a leading cause of invasive bacterial infections in newborns and is also responsible for diseases in older and immunocompromised adults. Prophages represent an important factor contributing to the genome plasticity and evolution of new strains. In the present study, prophage content was analyzed in human GBS isolates. Thirty-seven prophages were identified in genomes of 20 representative sequenced strains. On the basis of the sequence comparison, we divided the prophages into eight groups named A–H. This division also corresponded to the clustering of phage integrase, even though several different integration sites were observed in some relative prophages. Next, PCR method was used for detection of the prophages in 123 GBS strains from adult hospitalized patients and from pregnancy screening. At least one prophage was present in 105 isolates (85%). The highest prevalence was observed for prophage group A (71%) and satellite prophage group B (62%). Other groups were detected infrequently (1–6%). Prophage distribution did not differ between clinical and screening strains, but it was unevenly distributed in MLST (multi locus sequence typing) sequence types. High content of full-length and satellite prophages detected in present study implies that prophages could be beneficial for the host bacterium and could contribute to evolution of more adapted strains.     

Ⅱ型猪链球菌浙江分离株cps2J、ef、mrp基因的克隆及序列分析

目的阐明浙江省猪链球菌分离株的血清型分型,分析其与国内外其他猪链球菌Ⅱ型（SS2）菌株 cps2J、ef、mrp毒力基因的差异。方法参照GenBank上已发表的cps2J、ef、mrp毒力基因序列,设计引物 ,对6株浙江省猪链球菌分离株进行cps2J、ef、mrp全基因克隆及序列测定,并与国内外其他分离株的基 因序列进行比较。结果cps2J、ef、mrp基因的完整开放阅读框（ORF）分别为999bp、2532bp、3771bp,各 自编码333、843、1256个氨基酸。经对cps2J、ef、mrp毒力基因的序列比较与系统进化分析,6株浙江分 离株均分布在SS2发生群中,与国内外其他SS2菌株cps2J、ef、mrp基因核苷酸的同源性均较高,分别为 98.39%～100.0%、99.6%～100.0%和99.4%～100.0%,分离的年代和地区对基因的同源性和系统进化分支影 响不大。结论6株浙江省猪链球菌分离株为2型菌株,cps2J、ef、mrp基因序列非常保守,与GenBank上国 内及欧洲SS2菌株中相应序列有很高的同源性,可能有着相同的起源。     

Purulent Pericarditis in Sickle Cell Disease Due to Streptococcus agalactiae; a Unique Case Report and Literature Review

Purulent pericarditis is a localized infection with a thick, fibrinous hypercellular exudate and is historically associated with a high mortality. We describe a case of purulent pericarditis due to Streptococcus agalactiae (S. agalactiae) in a 30-year-old woman with sickle cell disease who presented with fever, dyspnea, and S. agalactiae septicemia. Despite timely initiation of antibiotics, she developed a large purulent pericardial effusion requiring surgical pericardiocentesis followed by a pericardial window. At 14?months follow-up, she has remained asymptomatic without sequelae. A review of the literature contained only four patients with purulent pericarditis in sickle cell patients. We discuss the unique aspects of this case in the context of purulent pericarditis in the age of modern antibiotics and hypothesize on the pathogenesis of delayed pericardial effusion after pericarditis.     

Streptococcus agalactiae pyomyositis in a patient with primary biliary cholangitis: A case report

Pyomyositis is an uncommon clinical scenario; it is usually associated with predisposing factors, including poorly controlled diabetes mellitus, trauma history, and immunocompromise. We discuss the case of an elderly woman with a 20-year history of diabetes mellitus and remissive breast cancer after modified radical mastectomy and subsequent chemotherapy 28 years previously. The patient presented with severe shoulder pain and gradual swelling. After examination, pyomyositis was diagnosed and debridement surgery was performed. Culture of the wound samples showed the growth of Streptococcus agalactiae. During hospitalization, primary biliary cholangitis (PBC) was diagnosed incidentally, accompanied by poor glycemic control. After treatment with antibiotics for pyomyositis and ursodeoxycholic acid for PBC, the infection resolved in 8 weeks, and her glycemic control was improved after PBC treatment. It is possible that the long-term untreated PBC worsened insulin resistance and aggravated diabetes mellitus in this patient. To the best of our knowledge, this is the first reported case of pyomyositis caused by an unusual pathogen, S. agalactiae, in a patient with newly diagnosed PBC.