框镜鲤原代细胞培养和锦鲤疱疹病毒的分离鉴定
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摘要
锦鲤疱疹病毒病(KHVD)的暴发给鲤鱼养殖业造成了严重的经济损失,KHVD有效疫苗的研制迫在眉睫,锦鲤疱疹病毒(KHV)的分离是预防和控制本病的关键,而建立敏感细胞系是分离KHV的前提。本文以框镜鲤(Cyprinus carpio var. specularis)为实验材料,取吻端和尾鳍组织,采用组织块培养方法进行体外培养,建立和优化框镜鲤细胞体外培养的最佳条件,研究框镜鲤吻端和尾鳍组织在体外培养环境下的部分细胞特性,并分别将其传代细胞命名为MSC和MFC。同时,利用培养的MSC和MFC进行了KHV的分离。
框镜鲤吻端和尾鳍组织在不同培养基(MEM、DMEM、RPMI1640、M199)、血清浓度(5%、10%、15%、20%)和温度(20℃、26℃、30℃、37℃)下启动原代培养,通过对细胞迁出距离绘制生长曲线以及细胞迁出距离的均值间多重比较,结果表明,含有20%血清的M199,在26℃～30℃下培养,最适合吻端和尾鳍组织细胞的迁出和增殖。两种组织,培养3d即可有细胞迁出,并逐渐在组织块周围形成细胞单层,即生长晕,一个月左右细胞单层可长满70～80%,可用于传代培养。MSC和MFC传至第3代时可适应体外环境,且MSC活性好于MFC细胞；H.E.染色发现,MSC细胞着色比MFC细胞深,MSC多为纤维样细胞,而MFC多为上皮样细胞,两种细胞增殖迅速,均具有建立连续细胞系的潜力；以10%DMSO为冻存保护剂,分别冻存于-80℃和液氮中,结果显示液氮冻存细胞的复苏存活率低于-80℃冻存,MFC的存活率略高于MSC。通过病毒敏感性试验表明SVCV可以感染MSC和MFC,产生典型的CPE,并且可以显著提高病毒滴度。
本文利用上述培养的MSC和MFC细胞进行KHV分离,通过将正常细胞单层与患病鱼的肾细胞共培养,5-6d后出现典型的CPE,即细胞体积增大并出现细胞质空泡化；在电镜下可观察到典型的KHV病毒粒子,命名为KHV-cj;应用PCR方法扩增获得ORF25基因的849bp DNA片段,连接至pMD18-T载体中,经酶切鉴定后序列分析表明,与GenBank登录的三株KHV同源性均为99%,基因进化比较显示,与KHV-J株关系较近。将病毒培养物(TCID50效价为10-6.75)腹腔注射接种实验鱼可使其100%发病,并且症状和病理变化与自然感染KHV的鱼一致,死亡率为75%。表明本研究在国内首次分离到KHV。
The Kio herpesvirus disease has caused severe financial losses to fish breeders. Isolation of KHV was the key of prevent and control this viral disease. And the susceptive cells line was the antecedent of isolating KHV. Cyprinus carpio var. specularis were chosen for experimental materials in this study. Snout and tail fin were taken to culture by tissue explant in vitro. In order to found and optimite culture conditions for the both cells, and get some cell characteristics of MSC and MFC in vitro. And we isolate the KHV using the MSC or MFC.
The snout and tail fin were cultured under different mediun (MEM、DMEM、RPMI1640、M199), serum concentration (5%、10%、15%、20%), temperature(20℃、26℃、30℃、37℃).Drawed the growth curve and paired comparison the means of the distance of cells migration by LSD method, the result showed that snout and tail fin cells were most quickly multiplied under 26℃～30℃,20% serum concentration, M199.The cell migrated from snout and tail fin after cultured 3 days, and formed the outgrowth. After about a month, the cell monolayer could be subcultured. The 3rd generations could adapt the vitro conditions. The MSC were moer quickly multiplied than MFC. The MFC were like epithelial cells, deying light by H.E., but the MSC were fiber-like cell. They had the potential ability to become the tissue source of establishing continuous cell line. The both cells were cryopreserved with 10% DMSO in -80℃and liquid nitrogen. The result show that the survival rate of the cell cryopreserved in liquid nitrogen was lower than in -80℃,and MFC were higher than MSC. Both cells were sensitive with SVCV, could be observed CPE, and the virus titer increased markedly.
Kio herpesvirus was isolated from the infected carp in order to research and prevent Kio herpesvirus disease. The virus was isolated by co-cultivation of MFC (or MSC) monolayers with cells taken from kidneys of infected living fish. Cytopathic effects (CPE) were observed 5-6 days post co-cultivation of fresh MFC with the kidney cell.Virus induced formation of endoplasmic vacuoles in the infected cells. The KHV particles were reviewed under electron microscope by negatively stained the sediment of virus culture with 0.5% phosphotungstate. A 849bp DNA fragment of ORF25 gene was ampliped by PCR. The PCR product was cloned and sequenced. It was the same homology of 99.9% with three strain of KHV in GenBank. The phylogenetic tree analysis indicated that there was a close genetic relationship between KHV-cj and KHV-J. The symptom and pathological of the fish that were infected by intraperitoneal injected with lml virus culture(TCID50=10-6.75) was the same with natural infection. The morbility and mortality were 100% and 75%, respectively. This study demonstrated that we isolated for the first time KHV from the naturally infected fish in Chian.
框镜鲤吻端和尾鳍组织在不同培养基(MEM、DMEM、RPMI1640、M199)、血清浓度(5%、10%、15%、20%)和温度(20℃、26℃、30℃、37℃)下启动原代培养,通过对细胞迁出距离绘制生长曲线以及细胞迁出距离的均值间多重比较,结果表明,含有20%血清的M199,在26℃～30℃下培养,最适合吻端和尾鳍组织细胞的迁出和增殖。两种组织,培养3d即可有细胞迁出,并逐渐在组织块周围形成细胞单层,即生长晕,一个月左右细胞单层可长满70～80%,可用于传代培养。MSC和MFC传至第3代时可适应体外环境,且MSC活性好于MFC细胞；H.E.染色发现,MSC细胞着色比MFC细胞深,MSC多为纤维样细胞,而MFC多为上皮样细胞,两种细胞增殖迅速,均具有建立连续细胞系的潜力；以10%DMSO为冻存保护剂,分别冻存于-80℃和液氮中,结果显示液氮冻存细胞的复苏存活率低于-80℃冻存,MFC的存活率略高于MSC。通过病毒敏感性试验表明SVCV可以感染MSC和MFC,产生典型的CPE,并且可以显著提高病毒滴度。
本文利用上述培养的MSC和MFC细胞进行KHV分离,通过将正常细胞单层与患病鱼的肾细胞共培养,5-6d后出现典型的CPE,即细胞体积增大并出现细胞质空泡化；在电镜下可观察到典型的KHV病毒粒子,命名为KHV-cj;应用PCR方法扩增获得ORF25基因的849bp DNA片段,连接至pMD18-T载体中,经酶切鉴定后序列分析表明,与GenBank登录的三株KHV同源性均为99%,基因进化比较显示,与KHV-J株关系较近。将病毒培养物(TCID50效价为10-6.75)腹腔注射接种实验鱼可使其100%发病,并且症状和病理变化与自然感染KHV的鱼一致,死亡率为75%。表明本研究在国内首次分离到KHV。
The Kio herpesvirus disease has caused severe financial losses to fish breeders. Isolation of KHV was the key of prevent and control this viral disease. And the susceptive cells line was the antecedent of isolating KHV. Cyprinus carpio var. specularis were chosen for experimental materials in this study. Snout and tail fin were taken to culture by tissue explant in vitro. In order to found and optimite culture conditions for the both cells, and get some cell characteristics of MSC and MFC in vitro. And we isolate the KHV using the MSC or MFC.
The snout and tail fin were cultured under different mediun (MEM、DMEM、RPMI1640、M199), serum concentration (5%、10%、15%、20%), temperature(20℃、26℃、30℃、37℃).Drawed the growth curve and paired comparison the means of the distance of cells migration by LSD method, the result showed that snout and tail fin cells were most quickly multiplied under 26℃～30℃,20% serum concentration, M199.The cell migrated from snout and tail fin after cultured 3 days, and formed the outgrowth. After about a month, the cell monolayer could be subcultured. The 3rd generations could adapt the vitro conditions. The MSC were moer quickly multiplied than MFC. The MFC were like epithelial cells, deying light by H.E., but the MSC were fiber-like cell. They had the potential ability to become the tissue source of establishing continuous cell line. The both cells were cryopreserved with 10% DMSO in -80℃and liquid nitrogen. The result show that the survival rate of the cell cryopreserved in liquid nitrogen was lower than in -80℃,and MFC were higher than MSC. Both cells were sensitive with SVCV, could be observed CPE, and the virus titer increased markedly.
Kio herpesvirus was isolated from the infected carp in order to research and prevent Kio herpesvirus disease. The virus was isolated by co-cultivation of MFC (or MSC) monolayers with cells taken from kidneys of infected living fish. Cytopathic effects (CPE) were observed 5-6 days post co-cultivation of fresh MFC with the kidney cell.Virus induced formation of endoplasmic vacuoles in the infected cells. The KHV particles were reviewed under electron microscope by negatively stained the sediment of virus culture with 0.5% phosphotungstate. A 849bp DNA fragment of ORF25 gene was ampliped by PCR. The PCR product was cloned and sequenced. It was the same homology of 99.9% with three strain of KHV in GenBank. The phylogenetic tree analysis indicated that there was a close genetic relationship between KHV-cj and KHV-J. The symptom and pathological of the fish that were infected by intraperitoneal injected with lml virus culture(TCID50=10-6.75) was the same with natural infection. The morbility and mortality were 100% and 75%, respectively. This study demonstrated that we isolated for the first time KHV from the naturally infected fish in Chian.
引文
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