基因枪轰击不同剂量小鹅瘟病毒VP3基因疫苗在雏鹅体内的动态分布

本文开展了检测小鹅瘟病毒(GPV)VP3基因的实时荧光定量PCR(FQ-PCR)方法的建立和基因枪轰击不同剂量(1、3和6μg)GPV-VP3基因疫苗(pcDNA-GPV-VP3)在30日龄四川白鹅体内(心、肝、脾、肺、肾、法氏囊、胸腺、哈氏腺、十二指肠、空肠、回肠、直肠、盲肠、胰腺、血液、脑及注射部位皮肤)分布规律的研究。结果表明:①建立的FQ-PCR特异性强、灵敏度高、重复性好,核酸模板数与FQ-PCR测定的Ct值相关系数达到0.999,具有很好的直线相关性;②pcDNA-GPV-VP3各剂量免疫雏鹅1 h即可在各组织中检测到,其中注射部位含量最高,肝、肾、淋巴器官(脾、法氏囊、胸腺、哈氏腺)含量较高;③到免疫后217 d时,1μg组免疫雏鹅各个组织器官内仍检测到pcDNA-GPV-VP3的存在,但多数组织器官中的含量比1 h时约少了4个数量级,其中免疫部位减少了7个数量级;④血液中pcDNA-GPV-VP3的含量较少,且免疫后1 h~217 d各时间点的差异不显著(P≥0.05);⑤不同剂量pcDNA-GPV-VP3免疫雏鹅各组织中的含量呈现的总体规律为6μg组>3μg组>1μg组,但差异不显著(P≥0.05)。因此,FQ-PCR是定量检测pcDNA-GPV-VP3在免疫雏鹅体内含量的可靠方法,pcDNA-GPV-VP3免疫雏鹅后1 h时可分布至雏鹅体内各组织器官中并持续存在217 d以上。     

Effect of cell mediated immunity regulation of duck enhanced by duck IFN-α eukaryon expression plasmid and inoculated with DPV attenuated vaccine by gene-gun

In order to study the effect of cell mediated immunity regulation of duck IFN-α eukaryon expression plasmid (pcDNA-SDIFN-α) on duck plague virus (DPV) attenuated vaccine in ducks, pcDNA-SDIFN-α was administered to 28-day-old ducks at doses of 1, 3 and 6 μg per duck, respectively, by gene-gun. PBS and empty vector pcDNA were used as control. Fifteen days later, all ducks were injected with DPV attenuated vaccine and blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63 and 84 days after injection. T-lymphocyte proliferation tests (MTT) were used to detect the T-lymphocyte proliferation in the peripheral blood (PBL) of ducks. Blood samples collected at 7, 14, 21, 28, 35 and 49 days after injection were detected by fluorescence-activated cell sorter (FACS) for recording the number of CD₃ ⁺ T-lymphocytes of ducks. Results were as follows: (1) Reaction of T-lymphocytes in PBL to ConA (OD value) of ducks treated with pcDNA-SDIFN-α was higher than that of PBS and pcDNA control groups in 3–84 days. There were highly significant differences between the 1 μg per duck group and the two control groups in 3–84 days (P ⩽ (0.01), between the 3 μg per duck group and the two control groups in 3–84 days (P ⩽ 0.01, P ⩽ 0.05), and between the 6 μg per duck group and the two control groups in 7–49 days (P ⩽ 0.01, P ⩽ 0.05). The significant difference was also present between the groups of 1, 3 and 6 μg per duck in 3–35 days (P ⩽ 0.05). However, there was no significant difference between the 3 and 6 μg per duck groups (P ⩾ 0.05). The pcDNA control group was higher than PBS control group, but no difference was detected (P ⩾ 0.05). (2) Change of the number of CD₃ ⁺ T-lymphocytes in ducks administered with different doses of pcDNA-SDIFN-α was higher than that of PBS and pcDNA control groups in 7–49 days. The change in the 1 μg per duck group was significantly higher than that in PBS and pcDNA control groups in 14–49 days (P ⩽ 0.01). There were significant differences between the 3 μg per duck group and the two control groups in 21–49 days (P ⩽ 0.01, P ⩽ 0.05) and between the 6 μg per duck group and the two control groups in 7–49 days (P ⩽ 0.01, P ⩽ 0.05). However, no significant differences among the groups of 1, 3, and 6 μg per duck groups (P ⩾ 0.05) and between the two control groups (P ⩾ 0.05) were found. The results indicated that pcDNA-SDIFN-α administered 15 days before injection of DPV-attenuated vaccine could significantly enhance cellular immunity induced by DPV-attenuated vaccine. pcDNA-SDIFN-α is an excellent DPV-attenuated vaccine molecular adjuvant and the best result can be obtained with the dose of 1 μg per duck of pcDNA-SDIFN-α inoculated by gene-gun. __________ Translated from Acta Veterinaria et Zootechnica Sinica, 2007, 38 (10): 1066–1071 [译自: 畜牧兽医学报]     

棉花基因枪茎尖转化研究

对棉花茎尖轰击转化的多种因素进行了分析,建立了较为完善的基因枪茎尖转化体系,使抗性转化率达到4.7％～9.4％,转化周期缩短至2～3个月,改进了抗性筛选程序,建立了4次筛选法,获得91株抗生素抗性植株;同时对部分植株目的基因的表达量进行了初步的遗传分析。结果表明,茎尖的轰击状态与抗性筛选程序对转化周期与转化率具有决定性作用。