Dairy cow laminitis causes severe economic losses and animal welfare issues in dairy farms globally. Current clinical interventions for laminitis remain limited in terms of safety and efficacy, which poses significant challenges to pasture-based productivity. Previous studies demonstrated that the activation of PI3K/Akt and NF-κB signaling pathways is a crucial mechanism promoting inflammatory damage and lamellar tissue degradation, providing potential targets for the development of novel therapeutic agents for treating dairy cow laminitis. Quercetin, an accessible and inexpensive flavonoid, exhibits diverse biological activities, including anti-inflammatory, antioxidant, and metabolic-modulatory effects. The present study aimed to elucidate the anti-inflammatory mechanism of quercetin in LPS-induced inflammatory response in dairy cow lamellar keratinocytes. Our results showed that quercetin at a concentration of 25 µM effectively downregulated the mRNA expression of inflammatory factors, including TNF-α, IL-6, IL-1β, CXCL-1, CXCL-6, COX-2, and iNOS in association with elevated expression of IL-10. Concurrently, quercetin inhibited the activation of the PI3K/Akt and NF-κB signaling pathways. Further experiments demonstrated that co-treatment with ammonium pyrrolidinedithiocarbamate (PDTC, an NF-κB inhibitor) significantly potentiated the inhibitory effect of quercetin on the protein expression of p-IκBα and p-p65. This result confirmed that quercetin exerts its anti-inflammatory effect by targeting and modulating the NF-κB signaling pathway. Additionally, Akt overexpression markedly alleviated quercetin-induced suppression of both p-IκBα/p-p65 protein expression and the mRNA transcription of downstream pro-inflammatory factors (TNF-α, IL-6, and IL-1β) but further increased quercetin-enhanced IL-10 expression. In contrast, combined Akt silencing and quercetin treatment further reduces the activation of the NF-κB signaling pathway. In conclusion, our findings demonstrate that quercetin alleviates the inflammatory response in LPS-induced dairy cow lamellar keratinocytes by modulating the PI3K/Akt/NF-κB signaling pathway. This study provides experimental evidence for the potential application of quercetin in the prevention and treatment of dairy cow laminitis.

Streptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant Escherichia coli DH5α-ORF4-GFP (targeting GBS scpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine’s protective efficacy, biosafety and regulatory effects via multi-dimensional assays. The optimal regimen was single immersion at 1.5 × 104 CFU/mL, with a maximum RPS of 73.13

Shiga toxin-producing E. coli (STEC) is a kind of crucial foodborne pathogen. At present, numerous studies have shown that phage can effectively eliminate foodborne pathogens as a novel antibacterial agent without affecting the nutrition and flavor of fresh food. This research aimed to investigate the biological characteristics and potential application in food products of a novel phage LTH02 for STEC. Host range determination based on single-layer plates, stability assay based on double-layer plates, lytic activity test, and whole genome analysis were utilized to evaluate the biological characteristics of phage LTH02. The results showed that phage LTH02 had a lysis rate of 85.00 % and displayed stability across pH levels ranging from 5 to 7 and temperatures between 4 degrees degrees C and 60 degrees degrees C. Besides, phage LTH02 inhibited STEC proliferation in a period of 30 h at an MOI of 102 and lacked antibiotic genes or virulence factors. The sterilization effect on the STEC-contaminated food was evaluated through bacterial counting. Phage LTH02 exhibited an effective elimination in the STEC-contaminated milk, raw beef, and lettuce at temperatures of 4 degrees degrees C and 24 degrees degrees C. The effect of phage LTH02 on food quality was evaluated through the measurement of pH, lactose content, TVB-N content, color difference, and sensory score of food. The results showed that phage LTH02 prevented the reduction of food quality caused by STEC. In conclusion, the phage LTH02 may serve as a potential antibacterial agent for controlling STEC in the food industry to enhance food safety and quality.

Lameness, primarily caused by claw lesions, leads to serious economic losses and animal welfare problems in dairy farms worldwide. Currently, its treatment approaches include topical application of nonsteroidal anti-inflammatory drugs (NSAIDs), improvement of the feeding environment, and therapeutic hoof trimming. However, long-term administration of NSAIDs may lead to adverse effects such as gastrointestinal complications and drug residue issues in dairy products. Recently, there was an increasing interest in the application of flavonoids in livestock management. Quercetin, a naturally abundant and cost-effective flavonoid, possesses multifunctional bioactivities, including anti-inflammatory, antioxidant, and metabolic-modulating functions. The purpose of this study was to evaluate the effect of dietary quercetin addition on claw lesions in dairy cows and to elucidate the possible mechanisms involved. In dairy cows with claw lesions, dietary supplementation with quercetin (100 mg/kg body weight) over 90 days suppressed NF-κB and PI3K/Akt signaling pathways, evidenced by inhibition of the phosphorylation of PI3K, Akt, p65, and IκBα, while downregulating their gene expression. Concomitantly, quercetin reduced the mRNA expression of inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), C-X-C motif chemokine ligand 1 (CXCL-1), C-X-C motif chemokine ligand 6 (CXCL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS)) in lamellar tissues of dairy cows with claw lesions. In conclusion, our findings indicate that dietary quercetin supplementation alleviates claw lesions and decreases the inflammatory response of lamellar tissues in dairy cows by modulating the PI3K/Akt and NF-κB signaling pathways. These results suggest that quercetin may serve as a natural anti-inflammatory candidate for the management of claw lesions in dairy cattle, offering new insights to improve hoof health and animal welfare in modern dairy pastures.

Fasciola gigantica can modulate host immune mechanisms through excretory–secretory products (ESP). As one of the components of ESP, it is unknown whether Abelson tyrosine protein kinase (Abl) is involved in parasite–host immune interaction. To investigate the immunoregulatory function of Abl in Fasciola gigantica, we cloned and expressed the Fasciola gigantica Abl protein and assessed its effect on specific immune functions of buffalo peripheral blood mononuclear cells (PBMCs). Recombinant F. gigantica Abelson tyrosine protein kinase (rFgAbl) was expressed in Escherichia coli. Western blot analysis was performed to assess the reactivity of anti-rFgAbl antibodies with rFgAbl, serum from F. gigantica-infected buffalo, and excretion and secretion products of F. gigantica. Immunohistochemical analysis was conducted to determine the localization of FgAbl in tissues from larval stages and adult worms of F. gigantica. Furthermore, immunofluorescence analysis was utilized to evaluate the binding ability of the rFgAbl protein to buffalo peripheral blood mononuclear cells (PBMCs), as well as to investigate the effects of varying concentrations of rFgAbl protein (5, 10, 20, 40, and 80 μg/mL) on the functional responses of PBMCs. Anti-rFgAbl antibodies specifically recognize rFgAbl, serum from buffalo infected with F. gigantica, and FgESP. rFgAbl is localized in the cecum and capsule of juvenile worms, as well as in the testis and viellaria of adult worms. Additionally, rFgAbl enhances cell proliferation, migration, nitric oxide (NO) production, and phagocytosis, while also increasing the transcription levels of cytokines (IFN-γ, IL-12, TNF-α, IL-4, IL-10, and TGF-β). The results indicate that rFgAbl can influence the immune function of PBMCs. Further investigation into the immunomodulatory properties of the rFgAbl protein will enhance our understanding of the immune interaction mechanisms between trematodes and their hosts.

试验旨在探究罗非鱼无乳链球菌(GBS)膜蛋白gap的免疫原性.试验提取GBS的DNA,利用PCR扩增出gap基因,并与表达载体pET-28a-SUMO构建重组质粒,转化至大肠杆菌BL21;经IPTG诱导、SDS-PAGE分析和Western blot检测确定其表达效果.采用纯化的gap蛋白与弗氏佐剂乳化腹腔注射免疫罗非鱼,以间接ELISA法检测血清效价并使用GBS进行攻毒试验.结果显示,PCR扩增出1 011 bp大小的单条带gap基因,与预期一致;重组载体pET-28a-gap在23℃、0.4 mmol/L IPTG、12 h条件下成功表达49.8 ku的可溶性蛋白;ELISA检测显示,重组蛋白gap能够产生较高的血清抗体效价水平,对GBS攻毒后的罗非鱼相对保护率为64.1%.研究表明,重组蛋白gap具有较好的免疫原性,为鱼类的链球菌病的预防提供新的理论依据.

[Objective]The purpose of this study was to detect the efficacy of RNA interference(RNAi)of chicken myostatin gene(MSTN)by Split-GFP bimolecular fluorescence complementary technique and to compare it with com-monly used detection methods in order to validate the effectiveness and availability of the Split-GFP bimolecular fluores-cence complementary technique in the assessment of RNAi efficacy.[Method]The three synthesized shRNA lentiviral cloning vectors(shRNA-a,shRNA-b and shRNA-c)were respectively transfected to HEK 293TGFP11-MSTN cells which sta-bly expressed the GFP11-MSTN fusion protein.The optimal shRNA lentiviral cloning vector was screened by real-time fluorescence quantitative PCR and packaged as lentivirus.Subsequently,the HEK 293TGFP11-MSTN cells were infected with the lentivirus,and mCherry positive(mCherry+)cells were screened using hygromycin B,and the RNAi efficiency was detected by real-time fluorescence quantitative PCR and Western Blotting.The obtained mCherry+ cells were trans-fected with a pcDNA3.1(+)-GFP1-10 plasmid,and the RNAi efficiency was assessed by fluorescence microscopy obser-vation and flow cytometry.[Result]All of three shRNA lentiviral vectors had extremely significant inhibitory effects on MSTN gene expression(P<0.01,the same below),among which Anti-MSTN shRNA-a lentiviral vector had the best inter-fering efficacy.Anti-MSTN shRNA-a lentivirus infected HEK 293TGFP11-MSTN cells at the optimal MOI=3,and the relative expression of the MSTN gene and the expression of the GFP11-MSTN fusion protein were inhibited under this condition.The obtained mCherry+ cells were re-transfected with pcDNA3.1(+)-GFP1-10 plasmid,and both fluorescence microsco-py observation and flow cytometry assay results showed that the number of GFP+ cells was decreased greatly,and the per-centage of GFP+cells was reduced from 31.1%to 11.5%.The results of Split-GFP assay were consistent with those of real-time fluorescence quantitative PCR and Western Blotting detections,indicating that Anti-MSTN shRNA-a lentivirus could inhibit GFP11-MSTN fusion protein expression,thus playing a RNAi role.[Conclusion]Anti-MSTN shRNA-a lentivirus has the highest interfering efficacy on MSTN gene,which extremely significantly down-regulates the expression of MSTN gene after infection of HEK 293TGFP11-MSTN cells,and the percentage of GFP+ cells in the cells is greatly decreased after re-transfection of pcDNA3.1(+)-GFP1-10 plasmid,which is consistent with the results of real-time fluorescence quantita-tive PCR and Western blotting detection results,confirming that Slipt-GFP bimolecular fluorescence complementary tech-nique is a dependable and visualized method for RNAi detection.

The present study aimed to evaluate whether the direct protein delivery of PhiC31 integrase through electroporation could induce the PhiC31-mediated recombination in chicken DF-1 cells. Here， prokaryotic expression and affinity chromatography purification was used to produce a small ubiquitin-related modifier （SUMO）-tagged His-PhiC31 fusion protein， and a SUMO proteinase was utilized to generate a natural PhiC31 protein by removing SUMO-His tag. The two types of PhiC31 protein were respectively incubated with pBCPB+ plasmid in tubes to induce intermolecular recombination. Subsequently， a landing pad （LP） plasmid harboring the attP TT -DsRed2-attP CT fragment was electroporated into the chicken DF-1 cells， followed by another round of electroporation of the natural PhiC31 protein along with a donor plasmid carrying the attB TT -EGFP-HiBiT-attB CT segment. The results showed that a prokaryotic expression vector was successfully constructed， and SUMO-His-PhiC31 fusion protein was expressed in a resolvable pattern in E.coli with induction of isopropyl-β-D-thiogalactoside （IPTG）， and amount of the purified fusion protein reached 12 mg·L -1 ; both SUMO-His-PhiC31 and natural SUMO-free PhiC31 is capable of triggering the intermolecular recombination between attP and attB sites of pBCPB+ plasmid， and the recombinant efficiency of SUMO-His-PhiC31 is comparable to that of PhiC31 proteins （50% vs. 52%）; co-transfection of DF-1 cells with a PhiC31 expressing vector pCMVInt， LP and donor plasmids resulted in conversion of DsRed2 to EGFP， which was confirmed by fluorescent microscopy; the same results were observed when LP plasmid， the donor plasmid plus PhiC31 protein were successively electroporated to DF-1 cells， implying the occur of PhiC31-mediated recombination between attP TT and attB TT ， attP CT and attB CT ， respectively. The results indicated that the natural PhiC31 protein is biologically active. The protein is expected to serve as an important reagent in the Easi-CRISPR-TARGATT-mediated genome editing to generate transgenic chickens.

Laying hens are an excellent experimental oviduct model for studying reproduction biology. Because chicken oviduct epithelial cells (cOECs) have a crucial role in synthesizing and secreting ovalbumin, laying hens have been regarded an ideal bioreactor for producing pharmaceuticals in egg white through transgene or gene editing of the ovalbumin (OVA) gene. However, related studies in cOECs are largely limited because of the lack of immortalized model cells. In addition, the editing efficiency of conventional CRISPR-HDR knock-in in chicken cells is suboptimal (ranging from 1 to 10%) and remains elevated. Here, primary cOECs were isolated from young laying hens, then infected with a retrovirus vector of human telomerase reverse transcriptase (hTERT), and immortalized cOECs were established. Subsequently, an electroporation-based Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) method was adopted to integrate an EGFP-HiBiT cassette into the chicken OVA locus (immediately upstream of the stop codon). The immortalized cOECs reflected the self-renewal capability and phenotype of oviduct epithelial cells. This is because these cells not only maintained stable proliferation and normal karyotype and had no potential for malignant transformation, but also expressed oviduct markers and an epithelial marker and had a morphology similar to that of primary cOECs. EGFP expression was detected in the edited cells through microscopy, flow cytometry, and HiBiT/Western blotting. The EGFP-HiBiT knock-in efficiency reached 27.9% after a single round of electroporation, which was determined through genotyping and DNA sequencing. Two single cell clones contained biallelic insertions of EGFP-HiBiT donor cassettes. In conclusion, our established immortalized cOECs could act as an in vitro cell model for gene editing in chicken, and this electroporation-based Easi-CRISPR strategy will contribute to the generation of avian bioreactors and other gene-edited (GE) birds.

为了解广西部分地区猫隐孢子虫、十二指肠贾第虫和三毛滴虫的感染情况和基因型,试验采集南宁市、桂林市、柳州市、梧州市和河池市各宠物医院、流浪动物中心及送检的新鲜猫粪便样本300份,基于隐孢子虫SSU rRNA和gp60基因,十二指肠贾第虫bg、tpi和gdh基因及三毛滴虫ITS基因采用PCR方法对粪便DNA进行PCR扩增,阳性样本进行测序分析,构建遗传进化树.结果表明:共检测出22份隐孢子虫、24份十二指肠贾第虫和41份三毛滴虫阳性样本,阳性率分别为7.3％、8.0％和13.7％.幼猫三种原虫阳性率均显著高于成年猫(P＜0.05),腹泻猫的阳性率显著高于未腹泻猫(P＜0.05),雄性猫三毛滴虫的阳性率要显著高于雌性(P＜0.05).检测出的隐孢子虫都为猫隐孢子虫(Cryptosporidium felis,C.felis),根据gp60基因进行亚型分型,鉴定出XⅩⅨa和ⅩⅨc两种亚型.十二指肠贾第虫共鉴定出集聚体F、B和F+B三种基因型,其中集聚体F为优势基因型.说明广西部分地区猫普遍存在隐孢子虫、十二指肠贾第虫和三毛滴虫的感染,且感染的虫种和部分基因型为人兽共患型,由于宠物猫与人类的亲密接触,需要警惕这些肠道原虫在人和猫之间的互相传播,并采取相应措施来进行预防.

股骨干骨折多是由强烈的直接外力所导致,常伴有周围软组织损伤,引起患肢疼痛、肿胀和活动受限.手术固定可在短时间内减少疼痛并恢复运动功能.外固定支架由固定针、连接杆和固定夹组成,具有操作简便、切口小等优点,是兽医临床上较常见的骨折固定方法.论文报道了1例犬股骨骨折先行接骨板固定经历失败,继而用外固定支架联合髓内针固定术治疗过程.结果表明,第二次手术后,该犬患肢运动功能恢复良好.

BackgroundFasciola gigantica, a tropical liver fluke, infects buffalo in Asian and African countries, causing significant economic losses and posing public health threats. The diagnostic of buffalo fascioliasis caused by F. gigantica is vital in fascioliasis control and preventation. The 22nd gel filtration chromatography fraction of F. gigantica Excretory-Secretory Products (FgESP), namely Fasciola 22 (F22), which was used as a diagnostic antigen in indirect ELISA, has demonstrated great potential for fascioliasis diagnosing. In the absence of rapid diagnostic methods, the use of a colloidal gold immunochromatographic strip based on F22 was applied to detect F. gigantica infection in buffalo.MethodsIn the present study, the 22nd gel filtration chromatography fraction of FgESP (F22) was used as an antigen to establish the colloidal gold-based immunochromatographic strip (ICS). The nitrocellulose membrane was incubated with F22 at the test line (T line) and goat anti-mouse secondary antibody at the control line (C line). The mouse anti-buffalo secondary antibody 2G7 conjugated to colloidal gold particles was used as the detection system for line visualization. The strip was assembled and developed by optimizing reaction conditions. The sensitivity, specificity, stability, and early diagnostic value of the strip were evaluated employing buffalo-derived sera.ResultsAn immunochromatographic strip for the rapid detection of antibodies against F. gigantica-FgICS was developed. The strip demonstrated high sensitivity and specificity. Sensitivity tests confirmed positive results even when the positive reference serum was diluted 4,096 times. Except for one Schistosoma japonicum-positive serum that tested positive via FgICS, specificity tests confirmed no cross-reactivity with other positive sera of Schistosoma japonicum and Babesia bovis. The strip remained stable after storage at 4°C for up to 3 months. In infected buffalo, antibodies could be detected as early as 14–21 days post-infection. The detection of 17 positive sera yielded an 82.4% positive rate via FgICS vs. a 100.0% positive rate via ELISA based on FgESP. For FgICS, the 95% confidence interval of sensitivity was 84.8–95.4%, while specificity was 4.2–14.7%.ConclusionThe immunochromatographic strip FgICS developed in this study provides a simple and rapid method of F. gigantica antibody detection and infected buffalo monitoring in the field.

Cre recombinase is a widely-used genetic manipulation of genomic DNA. However, the conventional transfection of the DNA vectors expressing the Cre recombinase or viral transduction method yields low transfection efficiencies or insertion mutagenesis. The present paper evaluated whether the direct protein delivery of Cre recombinase through electroporation can induce the Cre-mediated recombination in the HEK 293T cells. Here, the small ubiquitin-related modifier (SUMO) -tagged His-Cre fusion protein was expressed in a soluble pattern in the Eschrichria coli (E.coli) cells, purified using affinity chromatography, and finally electroporated into the HEK 293T cells. These cells were previously transfected with three different Cre reporter vectors. The electroporation of the HEK 293T cells revealed either the activation of EGFP expression, or a decrease in RFP expression, and a concomitant increase in EGFP expression, indicating a desired recombinase-mediated cassette exchange (RMCE) event (conversion of RFP to EGFP), and a biological activity of the purified SUMO-His-Cre protein. The fusion protein is expected to serve in the Easi-CRISPR-LoxP-mediated genome editing to generate transgenic animal models.

[目的]克隆人γ干扰素(hIFNγ)基因,借助自剪切肽P2A,构建hIFNγ-P2A-RFP(红色荧光蛋白)融合基因转座子表达载体,并在HEK293T细胞中检测其表达水平.[方法]首先,以脂多糖(LPS)激活人外周血淋巴细胞,提取总RNA,经RT-PCR扩增hIFNγ基因;再通过重叠延伸PCR,获得hIFNγ-P2A-RFP融合基因,并将其插入PB002G转座子载体.经双酶切和DNA测序鉴定,将重组质粒转染HEK 293T细胞,荧光显微镜观察RFP表达,Western Blotting检测hIFNγ蛋白表达.[结果]克隆了hIFNγ基因,成功构建了PB-hIFNγ-P2A-RFP重组质粒;荧光显微镜观察发现RFP在HEK 293T细胞表达,转染效率达39.3％;Western Blotting检测显示,特异性蛋白条带约为21 kDa,与预期的hIFNγ蛋白大小一致.[结论]成功构建了人γ干扰素PB转座子真核表达质粒,该质粒在HEK 293T细胞中高效表达.

为研究水牛瘦素(Leptin)的功能,构建leptin基因真核表达载体EGFP-leptin,检测leptin在NIH/3T3细胞中的表达并分析其生物学特性.根据水牛leptin基因序列设计引物,并在上下游引物分别添加Hind Ⅲ和BamH Ⅰ酶切位点,以pMD18-T-leptin载体为模板,PCR扩增leptin基因,并亚克隆至载体pEGFP-Nl,以双酶切和DNA测序鉴定EGFP-leptin重组质粒;然后采用脂质体2000将重组质粒转染到NIH/3T3细胞,经G418筛选,获得稳定转染的细胞株,RT-PCR及Western blot检测EGFP-Leptin融合蛋白表达.动物试验分3组,处理组从小鼠尾静脉注入重组质粒1 μg/只,对照组和禁食组注射等体积生理盐水,3 d后称其体质量,采用ELISA法测定小鼠血清中Leptin水平.结果显示,成功构建真核表达载体EGFP-leptin,转染NIH/3T3细胞后出现绿色荧光;RT-PCR和Western blot进一步证实了 Leptin表达.动物试验显示,处理组和禁食组小鼠体质量下降速率高于对照组,差异显著(P＜0.05),处理组血清Leptin水平高于对照组,差异显著(P＜0.05).以上结果表明,EGFP-leptin能够在NIH/3T3细胞中稳定表达,产生的融合蛋白具有良好的生物学活性.

背主动脉注射是生产转基因鸡的经典方法,该方法不需换壳培养,但壳内注射技术难度大,并且无法实时观察后期胚胎发育情况.本实验对背主动脉注射法进行了改进,将增强型绿色荧光蛋白(EGFP)腺相关病毒(Adeno-associated Virus,AAV)壳外注射至150枚HH 14～16期(Hamburger-Hamilton Stage 14～16)鸡胚背主动脉中,再进行换壳培养,继续孵化至出壳,观察和分析改良背主动脉注射法与传统背主动脉注射法和胚盘下腔注射法对发育8、14、18、21 d鸡胚存活率、孵化率以及EGFP阳性检出率的影响.结果 表明:改良背主动脉注射组鸡胚存活率均高于传统背主动脉注射组与胚盘下腔注射组(P<0.05或P<0.01);改良背主动脉注射组的鸡胚孵化率(37％)高于传统背主动脉注射组(16％)与胚盘下腔注射组(28％)(P<0.01);荧光蛋白手电筒检测显示,改良背主动脉注射组鸡胚EGFP阳性率(17％)明显高于传统背主动脉注射组(13％)与胚盘下腔注射组(12％)(P<0.01).综上,背主动脉壳外注射结合换壳培养提高了转基因鸡胚胎孵化率和EGFP阳性检出率,对提高转基因鸡效率具有重要价值.

冷冻切片的优点是能完好保存有机溶剂及热敏感抗原,其缺点是制片过程中形成冰晶影响免疫组织化学检测结果.为了解决这一难题,我们对现有的冷冻切片法进行了改良.多聚甲醛固定和蔗糖脱水后的脑、肺和睾丸组织,在包埋和切片之前,利用OCT包埋剂(聚乙二醇和聚乙烯醇水溶性混合物)进行预包埋,切片后进行苏木精伊红染色(hematoxylin eosin staining,HE染色)和免疫组织化学染色(Immunohistochemical staining,IHC染色),并比较了改良冷冻切片法、常规冷冻切片法和石蜡切片法制作的切片HE和IHC染色效果.结果 显示,切片前使用OCT包埋剂充分浸润组织6～48 h,可显著地提高冷冻切片质量;与常规冷冻切片法相比,用改良法制作的睾丸和脑组织冷冻切片结构更加清晰,因冰晶形成的空洞较少,总体质量均得到极大提高;与传统石蜡切片相比,改良法制作的肺组织冷冻切片质量尤佳.本研究建立了一种适用于免疫组织化学研究的冷冻切片新方法,无需使用液氮骤冷,大幅降低了多组织样品制片的难度,并且获得了正确的IHC染色结果,从而简化了实验流程,为组织形态学研究和抗原检测提供了可靠手段.

Streptococcus agalactiae is a common pathogen in aquatic animals, especially tilapia, that hinders aquaculture development and leads to serious economic losses. Previously, a S. agalactiae strain named HN016 was identified from infected tilapia, and the attenuated strain YM001 was subsequently obtained by continuous passaging in Tryptic Soy Broth (TSB) medium. YM001 has been demonstrated as a safe vaccine for S. agalactiae infection in tilapia. To understand the molecular bases of the virulence of these two strains, we performed proteomic and transcriptomic analysis to reveal the protein and gene expression changes in the liver and intestine during the infection process. HN016 significantly decreased the contents of white blood cells (WBCs), neutrophils (NEUs), red blood cells (RBCs) and hematocrit (HCT) and increased the levels of total protein (TP), albumin (ALB) and globulin (GLO), while no such significant differences were observed when comparing the control with YM001. During the infection process, pathogenic peptidoglycan hydrolase, CSPA and membrane proteins were significantly differentially expressed between YM001 and HN016. Furthermore, both proteome and transcriptome data showed that the complement and coagulation cascades pathway and the antigen processing and presentation pathway were stimulated in the liver and intestine, respectively, by YM001 infection compared to HN016 infection. The interaction network analysis of key virulence genes from pathogens suggested that CSPA, as a key node, affects the expression of DOLPP1, MIPEP, PA2G4, OCIAD1, G3BP1 and CLIC5 with a positive correlation. The present evidence suggests that during the infection process, CSPA was the key genes contributing to low virulence in YM001.

Sox2 is known to play an important role in maintaining the totipotency and self-renewal of embryonic stem cells. The purpose of this study was to prepare an anti-chicken Sox2 polyclonal antibody using prokaryotic expression techniques, to evaluate its specificity and to use it to investigate the expression and distribution of Sox2 in the chicken brain and lungs. The chicken Sox2 gene was amplified and subcloned to a pET-30a vector to construct a prokaryotic expression vector, pET-Sox2. A His-Sox2 fusion protein was expressed, purified, and used to prepare an antichicken Sox2 polyclonal antibody. Western blotting revealed that the antichicken Sox2 antibody could specifically bind not only to the purified His-Sox2 fusion protein but also to the endogenous Sox2 protein in the testes of chicken, showing a distinct dose-dependent relationship between antigen and Sox2 antibody. Indirect immunofluorescent staining of Sox2-overexpressing cells showed strong nuclear and diffuse cytoplasmic immunoreactivity for Sox2 in the antichicken Sox2 antibody-staining cells. A CRISPR/Cas9 effector system-mediated Sox2 knockdown assay indicated that Sox2 expression in HEK 293T cells was downregulated in the presence of doxycycline but upregulated in the absence of doxycycline. In addition, cryosectioning and immunohistochemical staining illustrated that most spermatogonia in the seminiferous tubules, and a small number of Sertoli and Leydig cells, were positive for Sox2. The antichicken Sox2 antibody was also successfully used to investigate the expression and distribution of Sox2 in the chicken cerebellar cortex, optic tectum, cerebral cortex, and lungs. The results of this study confirmed the specificity of the antichicken Sox2 polyclonal antibody, which will be available for the study of biological functions of the chicken Sox2 gene and the self-renewal mechanisms of chicken pluripotent stem cells.