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通过构建牛病毒性腹泻病毒(BVDV)驼源重链抗体文库,并鉴定抗体文库的多样性,为筛选抗BVDV特异性重链抗体奠定基础。利用灭活的BVDV免疫双峰驼,多次免疫后测定血清抗体滴度,采集免疫后的血液并分离其外周淋巴细胞,抽提RNA,用RT-PCR法扩增重链抗体可变区(VHH)片段;将其克隆至载体pCANTAB5E,电转大肠杆菌TG1获得VHH抗体基因库,检测抗体库库容量,随机挑取克隆测序验证抗体文库多样性。结果表明,所构建的抗体文库的库容量为4.32×105;测序和聚类分析表明,抗体文库多样性丰富。利用辅助噬菌体救援后,得到噬菌体展示文库,测定滴度达1.3×1012 cfu/mL。 相似文献
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In order to study the defensive role and action mechanism of antibacterial peptide NK-Lysin during body reaction, when pathogen infect animal body. This study detected the expression of NK-Lysin induced by different concentrations of lipopolysaccharide(LPS) and in different immune organs by Real-time fluorescence quantitative PCR, and explored the generation and response of sheep antimicrobial peptide NK-Lysin in immune organs. The cloning vector of target gene NK-Lysin and reference gene GAPDH were constructed, drew the standard curve and detected the expression of NK-Lysin. The results showed that inducing with the LPS concentrations of 0.01, 0.1, 1 and 10 μg/mL, respectively,the expression of NK-Lysin reached the maximum within 8 h. There was no positive correlation between the expression of NK-Lysin and concentration of LPS. The expression of NK-Lysin mRNA had varying degrees in pre-shoulder lymph, spleen and blood. The expression in spleen was extremely significantly different compared with other organs(P＜0.01). Immune organs could express the antimicrobial peptide NK-Lysin in a relatively short time, and NK-Lysin participated in the body’s innate immunes responses. 相似文献
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试验旨在建立一套完整的绵羊甲状腺细胞的原代培养方法,为绵羊甲状腺功能的体外研究奠定基础。通过常规消化分离得到甲状腺滤泡上皮细胞,光镜下对不同培养时间的细胞进行形态学观察,并结合RT-PCR、细胞免疫荧光和ELISA,对培养的绵羊甲状腺细胞从形态,甲状腺球蛋白(TG)、甲状腺过氧化物酶(TPO)、促甲状腺激素受体(TSHR)特异基因mRNA表达,TG特异抗原蛋白表达和甲状腺激素T3、T4的分泌功能等方面进行鉴定。结果显示,绵羊甲状腺细胞在体外呈贴壁式生长,具有上皮样细胞特点;TG染色呈胞浆阳性,具有特异TPO、TG、TSHR mRNA表达及分泌T3、T4的功能,但T3、T4的分泌量随培养时间的延长呈递减趋势。本研究成功地建立了简便可行的绵羊甲状腺细胞的原代培养方法,为深入研究绵羊甲状腺的功能提供了试验平台。 相似文献
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