斑点叉尾鮰病毒（CCV）ORF6基因“自杀性”DNA疫苗的构建与免疫效果研究
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摘要
本研究构建了斑点叉尾鮰传统DNA疫苗和“自杀性”DNA疫苗,鉴定检测了两者在细胞中的表达差异,并对两种疫苗的免疫效果进行了比较。
1.斑点叉尾鮰病毒(Channel Catfish Virus, CCV)“自杀性”DNA疫苗的构建
根据斑点叉尾鮰病毒(CCV) ORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到“自杀性”DNA疫苗载体pSFV与常规DNA疫苗载体pcDNA3.1中,转化大肠杆菌DH5α后提取质粒,构建“自杀性”DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切以及序列测定等方法证实重组质粒构建正确。
2. CCV ORF6基因在细胞中的表达
将待验证的表达CCV ORF6基因的“自杀性”DNA疫苗和常规DNA疫苗重组质粒分别转染293T细胞(人胚肾细胞),间接免疫荧光试验表明ORF6基因在细胞中均获得表达,但“自杀性”DNA疫苗表达载体的表达量显著低于常规DNA疫苗表达载体。
3.CCV“自杀性”DNA疫苗对斑点叉尾鮰免疫指标的影响
用构建的“自杀性”DNA疫苗ps-ORF6(Ⅲ组)与常规DNA疫苗pcd-ORF6(IV组)两种质粒制作的疫苗及对照的另外三种免疫剂(Ⅰ组为“自杀性”空载体pSFV制成、Ⅱ组为常规载体pcDNA3.1 (+)制成、Ⅴ组为缓冲液PBS)免疫80±10g的斑点叉尾鮰,分别在第0周、第2周、第4周、第6周、第8周等不同时间取血检测血液白细胞吞噬活性、血清溶菌酶活性、血液总蛋白含量等指标。实验结果与分析如下：(1)CCV“自杀性”DNA疫苗引起的白细胞吞噬活性较对照组有显著性差异(P<0.05),均高于其它对照组。表明CCV“自杀性”DNA疫苗能够更好地增强鱼体白细胞吞噬活性,增强免疫力。本实验结果也表明,影响斑点叉尾鮰白细胞吞噬活性的因素较多,实验各组都有所增强。(2)Ⅰ、Ⅱ、Ⅲ、Ⅳ组血清溶菌酶含量比Ⅴ组高,其中Ⅲ组和Ⅳ组显著高于其它组别(P<0.05),同时,Ⅲ组显著高于Ⅳ组(P<0.05)。表明CCV“自杀性”DNA疫苗能够有效地提高斑点叉尾鮰血清溶菌酶含量。(3)各实验组均能引起斑点叉尾鮰血清蛋白含量的增加,但是从组间存在显著性差异来看,CCV“自杀性"DNA疫苗相对其它组没有明显的优势,其结果可能受到外界的影响与免疫试剂的影响有交叉共同作用。
4.CCV“自杀性”DNA疫苗对生长的影响。
统计免疫后各组斑点叉尾鮰的生长情况,发现各组生长情况良好,组间差异不显著,表明各组免疫剂对斑点叉尾鮰生长的影响不大。
In this study, channel catfish (Ictalurus punctatus), suicide DNA vaccine and conventional DNA vaccine were constructed and their expression levels in the cells were compared. The immunity effects were evaluated and compared after injection of conventional and suicide DNA vaccines. The main contents are as follows:
1. The construction of suicide DNA vaccine of Channel Catfish Virus (CCV) ORF6 gene The suitable primers were designed according to CCV ORF6 gene sequence. The gene was cloned into suicide DNA vaccine vector pSFV and conventional DNA vaccine vector pcDNA3.1 respectively. After transformation into E. coli DH5a and extraction plasmid, the suicide DNA vaccines ps-ORF6 and conventional DNA vaccine pcd-ORF6 were constructed. The methods of PCR, double enzymes digestion and DNA sequencing were used to confirm that CCV ORF6 gene was correctly inserted into the vector.
2. The expression of CCV suicide DNA vaccine in the cells. The recombinant plasmids were transfected into 293T cells(human embryo kidnet cell). Indirect immunofluorescence assay showed that both were expressed, but the express level of the suicide DNA vaccine was lower than that of conventional DNA vaccines.
3. The effects of CCV suicide DNA vaccine to immunity of channel catfish. The channel catfish from 70 g to 90 g was divided into five groups and immunized by intramuscular injection using the suicide DNA vaccines (group III) and conventional DNA vaccines (group IV) of channel catfish and the other three control groups were injected with other three reagents, the vector of pSFV for group I, the vector of pcDNA3.1 (+) for group II and PBS for group V. At the weeks of 0,2,4,6,8, the phagocytic activity of leucocytes and bactericidal activity of phagocytes were tested by using MTT dying method, lysozyme and protein were detected by using kit.
In the test, the significant difference was found between the suicide DNA vaccines and conventional DNA vaccines (P＜0.05), which were both higher than the three control groups. The results showed that the suicide DNA vaccines may enhance the phagocytic activity of leucocytes of the channel catfish. The results also indicated that the phagocytic activity of leucocytes of the channel catfish was influenced by various factors, as the phagocytic activities of leucocytes in all the groups have been upgraded. In the test of determine the lysozyme activity, the suicide DNA vaccines and conventional DNA vaccines had significant difference with the other control groups (P<0.05), and the value in the group III was significantly higher than that in the group IV (P<0.05). The results showed that the suicide DNA vaccines can improve the content of serum lysozyme to some extent. All the groups increased the total protein of the channel catfish, while the results showed the suicide DNA vaccines had no advantage and the environments did influence the value of the total protein.
4. The impacts of the suicide DNA vaccines on the growth performance of channel catfish
After the suicide DNA vaccines injection, the growth performance were evaluated during the following next days. Respectively, According to statistical analysis, there was no significant difference among the five groups (P>0.05).
1.斑点叉尾鮰病毒(Channel Catfish Virus, CCV)“自杀性”DNA疫苗的构建
根据斑点叉尾鮰病毒(CCV) ORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到“自杀性”DNA疫苗载体pSFV与常规DNA疫苗载体pcDNA3.1中,转化大肠杆菌DH5α后提取质粒,构建“自杀性”DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切以及序列测定等方法证实重组质粒构建正确。
2. CCV ORF6基因在细胞中的表达
将待验证的表达CCV ORF6基因的“自杀性”DNA疫苗和常规DNA疫苗重组质粒分别转染293T细胞(人胚肾细胞),间接免疫荧光试验表明ORF6基因在细胞中均获得表达,但“自杀性”DNA疫苗表达载体的表达量显著低于常规DNA疫苗表达载体。
3.CCV“自杀性”DNA疫苗对斑点叉尾鮰免疫指标的影响
用构建的“自杀性”DNA疫苗ps-ORF6(Ⅲ组)与常规DNA疫苗pcd-ORF6(IV组)两种质粒制作的疫苗及对照的另外三种免疫剂(Ⅰ组为“自杀性”空载体pSFV制成、Ⅱ组为常规载体pcDNA3.1 (+)制成、Ⅴ组为缓冲液PBS)免疫80±10g的斑点叉尾鮰,分别在第0周、第2周、第4周、第6周、第8周等不同时间取血检测血液白细胞吞噬活性、血清溶菌酶活性、血液总蛋白含量等指标。实验结果与分析如下：(1)CCV“自杀性”DNA疫苗引起的白细胞吞噬活性较对照组有显著性差异(P<0.05),均高于其它对照组。表明CCV“自杀性”DNA疫苗能够更好地增强鱼体白细胞吞噬活性,增强免疫力。本实验结果也表明,影响斑点叉尾鮰白细胞吞噬活性的因素较多,实验各组都有所增强。(2)Ⅰ、Ⅱ、Ⅲ、Ⅳ组血清溶菌酶含量比Ⅴ组高,其中Ⅲ组和Ⅳ组显著高于其它组别(P<0.05),同时,Ⅲ组显著高于Ⅳ组(P<0.05)。表明CCV“自杀性”DNA疫苗能够有效地提高斑点叉尾鮰血清溶菌酶含量。(3)各实验组均能引起斑点叉尾鮰血清蛋白含量的增加,但是从组间存在显著性差异来看,CCV“自杀性"DNA疫苗相对其它组没有明显的优势,其结果可能受到外界的影响与免疫试剂的影响有交叉共同作用。
4.CCV“自杀性”DNA疫苗对生长的影响。
统计免疫后各组斑点叉尾鮰的生长情况,发现各组生长情况良好,组间差异不显著,表明各组免疫剂对斑点叉尾鮰生长的影响不大。
In this study, channel catfish (Ictalurus punctatus), suicide DNA vaccine and conventional DNA vaccine were constructed and their expression levels in the cells were compared. The immunity effects were evaluated and compared after injection of conventional and suicide DNA vaccines. The main contents are as follows:
1. The construction of suicide DNA vaccine of Channel Catfish Virus (CCV) ORF6 gene The suitable primers were designed according to CCV ORF6 gene sequence. The gene was cloned into suicide DNA vaccine vector pSFV and conventional DNA vaccine vector pcDNA3.1 respectively. After transformation into E. coli DH5a and extraction plasmid, the suicide DNA vaccines ps-ORF6 and conventional DNA vaccine pcd-ORF6 were constructed. The methods of PCR, double enzymes digestion and DNA sequencing were used to confirm that CCV ORF6 gene was correctly inserted into the vector.
2. The expression of CCV suicide DNA vaccine in the cells. The recombinant plasmids were transfected into 293T cells(human embryo kidnet cell). Indirect immunofluorescence assay showed that both were expressed, but the express level of the suicide DNA vaccine was lower than that of conventional DNA vaccines.
3. The effects of CCV suicide DNA vaccine to immunity of channel catfish. The channel catfish from 70 g to 90 g was divided into five groups and immunized by intramuscular injection using the suicide DNA vaccines (group III) and conventional DNA vaccines (group IV) of channel catfish and the other three control groups were injected with other three reagents, the vector of pSFV for group I, the vector of pcDNA3.1 (+) for group II and PBS for group V. At the weeks of 0,2,4,6,8, the phagocytic activity of leucocytes and bactericidal activity of phagocytes were tested by using MTT dying method, lysozyme and protein were detected by using kit.
In the test, the significant difference was found between the suicide DNA vaccines and conventional DNA vaccines (P＜0.05), which were both higher than the three control groups. The results showed that the suicide DNA vaccines may enhance the phagocytic activity of leucocytes of the channel catfish. The results also indicated that the phagocytic activity of leucocytes of the channel catfish was influenced by various factors, as the phagocytic activities of leucocytes in all the groups have been upgraded. In the test of determine the lysozyme activity, the suicide DNA vaccines and conventional DNA vaccines had significant difference with the other control groups (P<0.05), and the value in the group III was significantly higher than that in the group IV (P<0.05). The results showed that the suicide DNA vaccines can improve the content of serum lysozyme to some extent. All the groups increased the total protein of the channel catfish, while the results showed the suicide DNA vaccines had no advantage and the environments did influence the value of the total protein.
4. The impacts of the suicide DNA vaccines on the growth performance of channel catfish
After the suicide DNA vaccines injection, the growth performance were evaluated during the following next days. Respectively, According to statistical analysis, there was no significant difference among the five groups (P>0.05).
引文
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