猪细小病毒PK-15细胞适应株的培育及增殖特性

为了研究猪细小病毒不同接毒方式的增殖规律及在不同细胞的病毒含量差异。本实验利用PK-15细胞对猪细小病毒(Porcine parvovirus,PPV)分离株进行适应性培养。针对PPV的NS1基因设计特异性引物,建立实时荧光定量PCR方法。利用该方法检测PPV分离株同步和分步接毒的病毒含量,绘制一步生长曲线;同时检测PPV在HeLa、MDBK、PK-15、ST、F81、BHK-21和Marc-145细胞上的增殖特性。结果显示,PPV分离株盲传至12代产生CPE,继续传代培养10代仍能产生稳定的细胞病变,成功培育出PK-15细胞适应株。一步生长曲线显示,分步接毒的病毒含量高于同步接毒,而增殖周期短于同步接毒;PPV在不同细胞的增殖结果显示,各细胞开始出现CPE的时间依次为PK-15、ST、HeLa和MDBK,病毒含量高低依次是ST、PK-15、MDBK和HeLa,而不能在F81、BHK-21和Marc-145细胞上增殖。本实验首次利用荧光定量PCR方法成功分析PPV不同接毒方式的增殖规律及不同细胞的增殖特性,为PPV基础研究和疫苗生产提供资料。     

IFN-γ对PCV-2增殖效果的影响

在病毒培养物中添加IFN-γ,研究提高PCV-2病毒滴度的最佳培养方法。以不同活性单位的IFN-γ和不同浓度的NH₄Cl配合使用分别添加至培养基,用免疫过氧化物酶细胞单层试验（IPMA）对病毒毒价（TCID₅₀）进行测定;于72 h后分别测定添加与不添加IFN-γ接毒细胞的细胞周期。结果显示,500 U/mL IFN-γ和75 mmol/L NH₄Cl处理PK-15后病毒的TCID₅₀达到最大值5.9;且无论在病毒接种细胞前还是接种后添加IFN-γ均大幅提高病毒增殖力,而且两者提高幅度相同;细胞周期测定显示,最适量的IFN-γ和NH₄Cl配合使用后,细胞增殖活性增加。说明,重组IFN-γ可以提高PCV-2在PK-15中的病毒增殖滴度。     

猪细小病毒核酸疫苗的构建及其对小鼠免疫原性的研究

将猪细小病毒VP2基因克隆至pCI-neo真核表达载体中,构建了pCIneo-VP2重组质粒,转染至PK-15细胞中,利用免疫荧光方法检测在体外表达情况;并以小鼠为动物模型,将pCIneo-VP2、pCI-neo重组质粒、猪细小病毒活疫苗和对照组通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-VP2质粒 1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-VP2能够有效诱导机体产生体液免疫和细胞免疫,为研制出高效、新型猪细小病毒疫苗提供了科学依据和试验依据。     

通过基因组定量研究猪瘟病毒在细胞中的增殖特性

应用间接免疫荧光、Real-time PCR和病毒感染滴度(TCID50)测定技术,分别从病毒抗原、病毒基因组RNA复制水平和病毒感染滴度变化3个方面,研究了猪瘟病毒(CSFV)在PK-15细胞中增殖的特点,用猪瘟病毒石门株感染96孔板培养的细胞,1×102个TCID50/孔,间接免疫荧光检测结果显示感染后8h能检测到被荧光抗体染色的感染细胞,随感染时间的延长,出现荧光的细胞数量逐渐增多,在感染后72h,几乎所有细胞均能出现荧光。Real-time PCR结果显示在细胞感染初期的8~24h,病毒的基因组RNA复制呈加速趋势,其拷贝数在感染后72h达到高峰。此外,在感染后8h能检测到病毒基因组负链RNA转录,不过负链RNA在病毒增殖过程中维持在较低的水平。TCID50测定结果表明CSFV的感染滴度增加趋势与基因组类似,在病毒感染8h后能检测到具有感染性的子代病毒,感染滴度在8~20h之间逐渐增长,24~48h之间增长速度稍减慢,在感染后48~52h达到高峰,能在72h之内维持较高的感染滴度。     

猪Mx1和牛Mx1蛋白在PK-15细胞中的表达及其对伪狂犬病病毒的抑制

目的:研究猪Mx1和牛Mx1蛋白在PK-15细胞中的表达并检测其是否对伪狂犬病病毒(PRV)具有抑制作用。方法:从IBRS-2细胞和MDBK细胞中分别调取猪Mx1和牛Mx1基因,并克隆到pc DNA3.1/myc-His(-)B,构建得到真核重组表达质粒,以脂质体转染的方法将其分别导入到PK-15细胞,从mRNA水平和蛋白质水平鉴定重组质粒在细胞内的表达情况,然后用细胞毒性试剂盒检测这两种蛋白是否对PK-15细胞具有毒性。之后,通过荧光定量PCR检测猪Mx1和牛Mx1在攻毒后不同时间、不同攻毒剂量的条件下对PRV的抑制情况,并观察100TCID50病毒攻击细胞72h后的病变程度。结果:成功克隆了猪Mx1和牛Mx1基因,经mRNA水平和蛋白质水平证实,两种重组质粒在PK-15细胞内能够正常表达。从荧光定量PCR和细胞病变的角度来看,细胞内表达的Mx1蛋白对PRV具有显著性的抑制(P0.001)。结论:猪Mx1和牛Mx1基因在PK-15细胞中表达的Mx1蛋白能够抑制PRV在胞内的复制。     

猪α干扰素重组腺病毒的构建及其抗口蹄疫病毒活性研究

本研究利用RT-PCR方法扩增猪α干扰素成熟蛋白基因后构建了重组腺病毒质粒pAd-poIFN-α,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化后获得了重组腺病毒rAd-poIFN-α。该重组腺病毒于HEK-293A细胞连续传代至20代滴度稳定,为107TCID50/mL。RT-PCR检测证明目的基因在mRNA水平上可有效表达;在PK-15细胞上可以检测到较强的抗猪口蹄疫病毒活性,从而为研究猪口蹄疫免疫防治新技术奠定了重要基础。     

猪α干扰素重组腺病毒的构建及其抗口蹄疫病毒活性研究

本研究利用RT-PCR方法扩增猪α干扰素成熟蛋白基因后构建了重组腺病毒质粒pAd-poIFN-a,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化后获得了重组腺病毒rAd-poIFN-α.该重组腺病毒于HEK-293A细胞连续传代至20代滴度稳定,为107 TCID50/mL.RT-PCR检测证明目的基因在mRNA水平上可有效表达;在PK-15细胞上可以检测到较强的抗猪口蹄疫病毒活性,从而为研究猪口蹄疫免疫防治新技术奠定了重要基础.     

猪CD151转基因PK-15细胞系构建及其对猪繁殖与呼吸综合征病毒的易感性

[目的]建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)易感的猪CD151转基因PK-15细胞系,研究CD151分子在PRRSV感染猪源细胞中的作用.[方法]用RT-PCR从猪肺泡巨噬细胞中扩增CD151全长cDNA,测序正确后克隆人真核表达载体pcDNA3;用重组载体pcDNA-CD151转染PK-15细胞,经G418抗性筛选获得转基因细胞系PK15-CD151,用RT-PCR和免疫荧光试验检测CD151表达;用VR-2332株PRRSV分别感染PK-15细胞、PK15-CD151细胞、MARC-145细胞和3D4-CD163细胞,定期观察细胞病变,用RT-PCR和免疫荧光试验检测病毒RNA基因组和病毒抗原,用半数组织细胞感染剂量测定病毒滴度.[结果]从猪巨噬细胞中克隆得序列正确的猪CD151 cDNA;从重组载体转染的PK-15细胞培养中筛选得G418抗性细胞克隆,并能正确表达猪CD151分子;在PRRSV感染后,PK15-CD151细胞虽然不表现明显的细胞病变,但能检测到病毒RNA基因组和病毒抗原,并能产生较高滴度的感染性病毒;该细胞系已在体外传30代以上,第10、20、30代细胞的PRRSV滴度无明显变化.[结论]猪CD151基因转染能使非易感PK-15细胞获得对PRRSV的易感性,提示猪CD151参与PRRSV感染猪源细胞.     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。     

粘虫核型多角体病毒在同源寄主细胞系内复制的研究

对MsNPV在同源寄主细胞系NEAU Ms 94 7311内复制进行了研究。结果表明 ,接种MsNPV后 72h ,细胞核内开始有成熟的多角体形成 ,病毒接种后 2 4 0h感染率最大 ,达 36.2 % ,至2 64h多角体完全成熟 ,达 2 5.0PIB/感染细胞。病毒增殖曲线表明 ,接毒后 2 4h在培养基中开始检测出MsNPV NOV ,2 4 0h滴度达最大值 ,为 6.32× 10 5TCID50 /mL。病毒连续传代至第 7代或第 8代以后 ,其感染率、病毒滴度及多角体产量均有显著下降。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).