水貂CDV的分离鉴定及其主要基因的克隆与免疫研究
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摘要
犬瘟热(Canine distemper，CD)系由副粘病毒科麻疹病毒属的犬瘟热病毒引起的一种急性、热性、高度接触性的传染病。该病的流行与传播严重地影响了经济动物养殖业、野生动物保护业、动物园观赏业和养犬业的持续发展。随着生态环境的变化及犬瘟热病毒(Canine distemper virus，CDV)对流行因素的适应，其自然感染宿主范围在不断扩大，加之CDV变异株的出现，目前犬瘟热的发病率和致死率均有所升高，流行趋势由散在发生趋势向群发趋势发展，临床症状也越来越复杂，其危害也越来越大。特别是国宝大熊猫等珍稀动物及猕猴等灵长类动物的CDV感染，使CDV的致病地位日益突出。最新研究证实CDV在体外可感染人的前破骨细胞，在破骨细胞内增殖，表明CDV的自然宿主可能已扩展到了人类，犬瘟热可能是犬传染给人的第二个病毒性传染病。
由于CDV呈世界性分布，自然感染宿主种类广泛，且可在大量的不相关动物种类间交叉感染。因此，消灭CDV几乎是不可能的，但可以利用疫苗进行主动免疫控制犬瘟热。目前常用的犬瘟热疫苗有CDV死毒苗、MV疫苗、CDV弱毒疫苗。但是CDV死苗和MV疫苗不能提供动物持久的保护力，而CDV弱毒疫苗的稳定性较差，接种后易受母源抗体的干扰，对某些免疫缺陷动物和野生动物不安全，具有散毒的危险，所以人们已开始研制新型疫苗，如核酸苗、基因工程亚单位苗、重组病毒苗等控制犬瘟热。本研究进行了以下几方面的工作，为水貂CD核酸疫苗的研制奠定基础。
1，水貂犬瘟热病毒的分离与鉴定从山东省青岛市某水貂养殖场疑似犬瘟热的病死貂的肝脏中分离出1株病毒，并进行了系统的鉴定。结果表明：该毒株接种于Vero传代细胞后出现了CDV典型而有规律的细胞病变；其理化特性与CDV相同；致细胞病变作用可被兔抗CDV阳性血清阻断；包涵体检查在细胞浆中可见圆形或椭圆形嗜酸性包涵体；间接免疫荧光试验表明接种病毒的BHK-21细胞出现特异性的亮绿色荧光，而正常细胞则未见绿色荧光；提取接种病料后的Vero细胞培养物中的总RNA进行RT-PCR反应，扩增出了324bp和1053bp的目的片段。证明该毒株为CDV，命名为CDVSJ。
2，犬瘟热病毒分离株附着蛋白(H)基因的克隆与序列分析对犬瘟热病毒水貂分离株CDVSJ株的H基因进行了RT-PCR扩增，扩增产物纯化后与pMD-18T Simple Vector连接，并转化至E．coli JM109感受态细胞中，成功地构建了重组克隆质粒pMD-18T-CDVH，并测定其序列。测序结果表明，CDVSJ株H基因由1596 bp组成，编码532个氨基酸，含有5个潜在的N-联糖基化位点，分别在76～78、318～320、349～351、383～385、514～516；含有12个半胱氨酸残基，分别位于66、81、115、211、223、304、309、317、417、493、502、529位。与01-2689、007Lm、2544Han95、25259、A75-17、American dog、black leopard、Liud、CDTaichung、Convac、Danish dog、Danish mink、DK91D、GreenIandic dog、Hamatsu、Javelina、KDK-1、LP、Onderstepoort、Snyder Hill、Tanu96、TN、Ueno和Yanaka毒株核苷酸序列同源性分别为90.7％、91.0％、91.9％、92.2％、92.0％、91.2％、91.8％、91.9％、90.9％、96.9％、92.0％、92.4％、92.0％、92.1％、90.9％、91.8％、91.1％、92.5％、96.6％、98.6％、91.1％、91.1％、91.0％和91.0％；推导的氨基酸序列同源性分别为89.9％、90.1％、91.4％、90.2％、91.9％、91.4％、91.9％、91.7％、91.2％、95.1％、91.4％、91.9％、91.6％、91.4％、90.4％、91.2％、90.8％、91.4％、94.6％、97.0％、90.8％、90.6％、91.0％和90.8％。与Onderstepoort、Convac、Hamatsu、A75／17、TN株相比，根据推导的氨基酸序列比较分析表明，犬瘟热病毒分离株CDVSJ具有5个潜在的N-联糖基化位点，与Onderstepoort疫苗株相比，在318Aa～320Aa、383Aa～385Aa处多2个N-联糖基化位点；与疫苗Convac株和野毒A75／17株相比，在530Aa～532Aa处少1个N-联糖基化位点；与Hamatsu株相比，在236Aa～238Aa、511Aa～513Aa和530Aa～532Aa处少3个N-联糖基化位点；与TN株相比，在236Aa～238Aa和511Aa～513Aa处少2个糖基化位点。聚类分析结果表明CDVSJ株与SnyderHill毒株的亲缘关系最近，二者与疫苗株Onderstepoort和Convac具有较高的同源性，组成一组，而与标准野毒强毒株Hamatsu的亲缘关系较远。
3，犬瘟热病毒分离株融合蛋白(F)基因的克隆与序列分析对犬瘟热病毒水貂分离株CDVSJ株的F基因进行了RT-PCR扩增，扩增产物纯化后与pMD-18T Simple Vector连接，并转化至E．coli JM109感受态细胞中，成功地构建了重组克隆质粒pMD-18T-CDVF，并测定其序列。测序结果表明，CDVSJ株F基因由1053 bp组成，编码351个氨基酸，含有9个半胱氨酸残基；与01-2601株、2544-Han95株、5804P株、25259株、A75-17株、DOGDK91C株、ONP株、PDV-2株和98-2646株相比，核苷酸序列同源性分别为95.4％、93.8％、93.9％、94.3％、94.5％、93.9％、97.3％、94.7％和94.1％；推导的氨基酸序列同源性分别为97.4％、96.6％、97.7％、98.0％、97.4％、97.4％、97.2％、98.0％和96.6％。抗原指数比较结果说明分离株CDVSJ与疫苗Onderstepoort株、强毒A75-17株在40-50位氨基酸间存在较大差异。与Onderstepoort株、A75／17株和PDV-2株相比，各个毒株F蛋白的半胱氨酸残基数目及其位置保守，均为9个。这说明CDVF蛋白基因是一个相对保守的结构蛋白基因。
4，犬瘟热病毒分离株F基因的真核表达将克隆至pMD-18T simple vector中的F基因片段亚克隆至pcDNA3.1(+)真核表达载体中，成功地构建出真核重组表达质粒pcDNA3.1-F和pcDNA3.1-SF。经脂质体介导法将真核重组表达质粒pcDNA3.1-F和pcDNA3.1-SF转染至BHK-21细胞中，通过ELISA法、间接荧光抗体技术和RT-PCR技术检测，证实目的基因在哺乳动物细胞中获得了表达。
5，真核重组表达质粒的免疫研究为研究真核重组表达质粒的免疫特性，将真核重组表达质粒pcDNA3.1-F和pcDNA3.1-SF肌注日本大耳白兔进行免疫，同时设置pcDNA3.1(+)、生理盐水阴性对照组和犬瘟热弱毒疫苗阳性对照组。间接ELISA法检测结果显示，免疫兔体内产生了针对CDV的特异性抗体，且抗体水平随着免疫次数和免疫剂量的增加而升高，重组质粒组体液免疫水平显著高于生理盐水组和空载体组，但不及弱毒疫苗对照组。淋巴细胞转化试验结果表明，重组质粒免疫组产生了细胞免疫，但低于弱毒疫苗组。
本研究从疑似犬瘟热病死貂肝脏中分离出1株犬瘟热病毒，并对水貂源犬瘟热病毒分离株的F基因和H基因进行了克隆与序列分析，丰富了CDV分子生物学研究内容；构建的CDV F基因真核重组表达质粒，在实验动物体内产生了特异性的体液免疫和细胞免疫。为进一步研究CDV F和H基因的免疫保护作用，研制诊断和预防水貂犬瘟热新型、高效的诊断试剂和DNA疫苗奠定了基础。
Canine distemper (CD), caused by canine distemper virus (CDV) which belongs to the Morbillivirusgenus of the Paramyxoviridae virus family, is a acute and highly contagious disease causing considerabledamages to the fur animal farming industry, threatening wildlife conservation and zoo animal. With thechanges in the environment and CDV adaptation to prevailing factors, CDV natural host's range expandsincessantly. In addition, the new CDV variants leading to the incidence and mortality rate of CD infectedanimals are increasing, and prevalent and clinical symptoms developed are more and more complicated atthe present time. Therefore, its harmful effects become more serious. In particular, when giant panda andmacaque and so forth quadrumana are infected CDV, CDV pathogenesis became protrusion. Latestresearch confirms that CDV can infect human prosoma osteoclast and that indicates CDV natural hostextends the possibility that it could infect human beings. It may be the second viral infection diseasepassing from dog to human beings.
Due to worldwide distribution of CDV and the widespread natural host and huge cross infection,eliminating CDV is almost impossible. However, a vaccine can be used as an active immunity to controlCD. Traditional attenuated vaccine has intrinsic shortcomings despite its important role in controlling theoccurrence of CD. In order to develop a new kind of safe and efficient CDV genetically engineeredvaccine, we have conducted the following works:
1. Isolation and Identification of Canine Distemper Virus from Mink
A strain of Canine Distemper Virus was isolated and identified systemically from the liver of a deadmink suspected of having Canine Distemper, at a mink farm in Qingdao, Shandong Province. The resultsindicated that typical and disciplinary CDV cell pathological changes appeared and the physical andchemical characteristics were similar to that of CDV after the virus strain had inoculated Vero cell. Thecytopathogenic effect can be interdicted by Rabbit anti-CDV positive serum. Rotundity and ellipseeosinophil inclusion body can be seen in cytoplasm during inclusion body examination. Indirectimmunofluorescent assay showed that BHK-21 cell inoculated the CDV resulting in idiosyncratic brightgreen fluorescence and that green fluorescence did not appeared in the normal cell. The total RNAextracted from Vero cell culture inoculated with the virus were amplified with Reversetranscription-polymerase chain reaction(RT-PCR), and two objective segments (324bp and 1053bp) wereobtained Thus, the virus strain was proved to be CDV and named as CDVSJ.
2. Cloning and Sequencing Analysis on hemagglutinin (H) genes of Isolated Canine Distemper Virus Strain
H gene of CDVSJ strain was amplified with RT-PCR, and the purified RT-PCR products werelinked with pMD-18T Simple Vector, then transferred to E.coli JM109 competent cell. The recombinedcloning plasmid pMD-18T-CDVH was created and sequenced. Sequencing result indicated that H genesection of CDVSJ strain was of a 1596bp fragment encoding 532 amino acids, which contained 5potential N-glycosylation positions located in 76Aa～78Aa, 318Aa～320Aa, 349Aa～351Aa, 383Aa～385Aa, 514Aa～516Aa; and also contained 12 cysteine residues located at 66, 81, 115, 211,223, 304,309, 317, 417, 493, 502, 529 positions. Compared with the nucleotide sequences of 01-2689, 007Lm,2544Han95, 25259, A75/17, American dog, black leopard, Liud, CDTaichung, Convac, Danish dog,Danish mink, DK91D,Greenlandic dog, Hamatsu, Javelina, KDK-1, LP, Onderstepoort, Snyder Hill,Tanu96, TN, Ueno and Yanaka strains, the homology is 90.7%, 91.0%, 91.9%, 92.2%, 92.0%, 91.2%,91.8%, 91.9%, 90.9%, 96.9%, 92.0%, 92.4%, 92.0%, 92.1%, 90.9%, 91.8%, 91.1%, 92.5%, 96.6%,98.6%, 91.1%, 91.1%, 91.0% and 91.0%, respectively. Derivative amino acids homology are 89.9%,90.1%, 91.4%, 90.2%, 91.9%, 91.4%, 91.9%, 91.7%, 91.2%, 95.1%, 91.4%, 91.9%, 91.6%, 91.4%,90.4%, 91.2%, 90.8%, 91.4%, 94.6%, 97.0%, 90.8%, 90.6%, 91.0% and 90.8%, respectively. Comparedwith Onderstepoort, Convac, Hamatsu, A75/17 and TN strains, CDVSJ strain contained 5 potentialN-glycosylation positions according to derivative amino acids sequence analysis. There were two moreN-glycosylation position at 318Aa～320Aaand383Aa～385Aa compared with the Onderstepoort vaccinestrain. One N-glycosylation positions, 530Aa～532Aa, were lacking compared with Convac and A75/17strains. Three N-glycosylation positions, 236Aa～238Aa, 511Aa～513Aa and 530Aa～532Aa, werelacking compared with Hamatsu strains.Two N-glycosylation position, 236Aa～238Aa and 511Aa～513Aa was lacking compared with TN strain. Clustering analysis results showed that CDVSJ strain isclosely related to the Snyder Hill strain. Sequence homologies are a close comparison to Onderstepoortand Convac, which form one group. However, the kinship between them and standard virulent strainHamatsu is far away.
3. Cloning and Sequence Analysis of fusion (F) Gene of Canine Distemper Virus Strain
F gene of CDVSJ strain was amplified with RT-PCR and the purified RT-PCR product was linkedto pMD-18T Simple Vector, then transferred to E.coli JM109 competent cell. The recombined cloningplasmid pMD-18T-CDVF was built and sequenced. Sequencing results indicated that F gene section ofCDVSJ strain was of a 1053bp fragment encoding 351 amino acids of which 9 were cysteine residues.Compared with the nucleotide sequences of 01-2601, 2544-Han95, 5804P, 25259, A75-17, DOGDK91C,ONP, PDV-2 and 98-2646strains, homology are 95.4%, 93.8%, 93.9%, 94.3%, 94.5%, 93.9%, 97.3%,94.7% and 94.1%, respectively. Derivative amino acids homology are 97.4%, 96.6%, 97.7%, 98.0%,97.4%, 97.4%, 97.2%, 98.0% and 96.6%, respectively. Antigen index comparative results showed that there are great differences in 40-50th amino acids between isolated CDVSJ and vaccine Onderstepoortand the virulent A75-17 strain.Compared with Onderstepoort, A75/17 and PDV-2 strains, the number andposition of cysteine residues of F protein in each strain was the same, being 9. That determines that CDVF gene is a comparative conservative structural protein gene.
4. Eukaryotic expression of Canine Distemper Virus F gene
Cloned F gene segment in pMD-18T simple vector was re-cloned to pcDNA3.1 (+) eukaryonexpression vector plasmid, forming eukaryon recombinant expression plasmid pcDNA3.1-CDVF andpcDNA3.1-CDVSF successfully. Eukaryon recombinant expression plasmid pcDNA3.1-CDVF andpcDNA3.1-CDVSF were transfected into BHK-21 cell by using lipoplast mediate method and has beenproved with ELISA, indirect fluorescent antibody technique and RT-PCR detection that the objectivegene was successfully expressed in mammalian cell.
5. Immunization test of Eukaryon recombinant expression plasmid
In order to study immunity characteristics of Eukaryon recombinant expression plasmid, Eukaryonrecombinant expression plasmid pcDNA3.1-F and pcDNA3.1-SF were injected intramuscularly intoJapanese albino rabbit. Meanwhile, pcDNA3.1 (+), normal saline negative control and CDV vaccinepositive were used as control. ELISA results showed that specificity antibody to CDV was produced inthe Japanese albino rabbit. Moreover antibody level increased with increasing immunizing times anddoses. The humoral immunity level of recombinant plasmid group was significantly higher than those ofnormal saline and pcDNA3.1 (+) control groups, however, but lower than that of attenuated vaccinegroup. Lymphocyte transformation test results showed that recombinant plasmid immunity groupproduced cellular immunity, but was lower than that of the attenuated vaccine group.
A strain of CDV was isolated from the liver of the suspect CD dead mink, and H and F gene ofCDVSJ strain were cloned and sequenced, providing useful information for molecular biological study ofthe virus. The recombined eukaryotic expression plasmid of CDV F protein gene was successfullyexpressed in the body of laboratory animal, producing both humoral and cell immunity. The results of thestudy provide fundamental information for the further study on the protective function of CDV H and Fgene, for the development of new type and efficient diagnostic reagent for diagnosis and prevention ofCD and DNA vaccine.
由于CDV呈世界性分布，自然感染宿主种类广泛，且可在大量的不相关动物种类间交叉感染。因此，消灭CDV几乎是不可能的，但可以利用疫苗进行主动免疫控制犬瘟热。目前常用的犬瘟热疫苗有CDV死毒苗、MV疫苗、CDV弱毒疫苗。但是CDV死苗和MV疫苗不能提供动物持久的保护力，而CDV弱毒疫苗的稳定性较差，接种后易受母源抗体的干扰，对某些免疫缺陷动物和野生动物不安全，具有散毒的危险，所以人们已开始研制新型疫苗，如核酸苗、基因工程亚单位苗、重组病毒苗等控制犬瘟热。本研究进行了以下几方面的工作，为水貂CD核酸疫苗的研制奠定基础。
1，水貂犬瘟热病毒的分离与鉴定从山东省青岛市某水貂养殖场疑似犬瘟热的病死貂的肝脏中分离出1株病毒，并进行了系统的鉴定。结果表明：该毒株接种于Vero传代细胞后出现了CDV典型而有规律的细胞病变；其理化特性与CDV相同；致细胞病变作用可被兔抗CDV阳性血清阻断；包涵体检查在细胞浆中可见圆形或椭圆形嗜酸性包涵体；间接免疫荧光试验表明接种病毒的BHK-21细胞出现特异性的亮绿色荧光，而正常细胞则未见绿色荧光；提取接种病料后的Vero细胞培养物中的总RNA进行RT-PCR反应，扩增出了324bp和1053bp的目的片段。证明该毒株为CDV，命名为CDVSJ。
2，犬瘟热病毒分离株附着蛋白(H)基因的克隆与序列分析对犬瘟热病毒水貂分离株CDVSJ株的H基因进行了RT-PCR扩增，扩增产物纯化后与pMD-18T Simple Vector连接，并转化至E．coli JM109感受态细胞中，成功地构建了重组克隆质粒pMD-18T-CDVH，并测定其序列。测序结果表明，CDVSJ株H基因由1596 bp组成，编码532个氨基酸，含有5个潜在的N-联糖基化位点，分别在76～78、318～320、349～351、383～385、514～516；含有12个半胱氨酸残基，分别位于66、81、115、211、223、304、309、317、417、493、502、529位。与01-2689、007Lm、2544Han95、25259、A75-17、American dog、black leopard、Liud、CDTaichung、Convac、Danish dog、Danish mink、DK91D、GreenIandic dog、Hamatsu、Javelina、KDK-1、LP、Onderstepoort、Snyder Hill、Tanu96、TN、Ueno和Yanaka毒株核苷酸序列同源性分别为90.7％、91.0％、91.9％、92.2％、92.0％、91.2％、91.8％、91.9％、90.9％、96.9％、92.0％、92.4％、92.0％、92.1％、90.9％、91.8％、91.1％、92.5％、96.6％、98.6％、91.1％、91.1％、91.0％和91.0％；推导的氨基酸序列同源性分别为89.9％、90.1％、91.4％、90.2％、91.9％、91.4％、91.9％、91.7％、91.2％、95.1％、91.4％、91.9％、91.6％、91.4％、90.4％、91.2％、90.8％、91.4％、94.6％、97.0％、90.8％、90.6％、91.0％和90.8％。与Onderstepoort、Convac、Hamatsu、A75／17、TN株相比，根据推导的氨基酸序列比较分析表明，犬瘟热病毒分离株CDVSJ具有5个潜在的N-联糖基化位点，与Onderstepoort疫苗株相比，在318Aa～320Aa、383Aa～385Aa处多2个N-联糖基化位点；与疫苗Convac株和野毒A75／17株相比，在530Aa～532Aa处少1个N-联糖基化位点；与Hamatsu株相比，在236Aa～238Aa、511Aa～513Aa和530Aa～532Aa处少3个N-联糖基化位点；与TN株相比，在236Aa～238Aa和511Aa～513Aa处少2个糖基化位点。聚类分析结果表明CDVSJ株与SnyderHill毒株的亲缘关系最近，二者与疫苗株Onderstepoort和Convac具有较高的同源性，组成一组，而与标准野毒强毒株Hamatsu的亲缘关系较远。
3，犬瘟热病毒分离株融合蛋白(F)基因的克隆与序列分析对犬瘟热病毒水貂分离株CDVSJ株的F基因进行了RT-PCR扩增，扩增产物纯化后与pMD-18T Simple Vector连接，并转化至E．coli JM109感受态细胞中，成功地构建了重组克隆质粒pMD-18T-CDVF，并测定其序列。测序结果表明，CDVSJ株F基因由1053 bp组成，编码351个氨基酸，含有9个半胱氨酸残基；与01-2601株、2544-Han95株、5804P株、25259株、A75-17株、DOGDK91C株、ONP株、PDV-2株和98-2646株相比，核苷酸序列同源性分别为95.4％、93.8％、93.9％、94.3％、94.5％、93.9％、97.3％、94.7％和94.1％；推导的氨基酸序列同源性分别为97.4％、96.6％、97.7％、98.0％、97.4％、97.4％、97.2％、98.0％和96.6％。抗原指数比较结果说明分离株CDVSJ与疫苗Onderstepoort株、强毒A75-17株在40-50位氨基酸间存在较大差异。与Onderstepoort株、A75／17株和PDV-2株相比，各个毒株F蛋白的半胱氨酸残基数目及其位置保守，均为9个。这说明CDVF蛋白基因是一个相对保守的结构蛋白基因。
4，犬瘟热病毒分离株F基因的真核表达将克隆至pMD-18T simple vector中的F基因片段亚克隆至pcDNA3.1(+)真核表达载体中，成功地构建出真核重组表达质粒pcDNA3.1-F和pcDNA3.1-SF。经脂质体介导法将真核重组表达质粒pcDNA3.1-F和pcDNA3.1-SF转染至BHK-21细胞中，通过ELISA法、间接荧光抗体技术和RT-PCR技术检测，证实目的基因在哺乳动物细胞中获得了表达。
5，真核重组表达质粒的免疫研究为研究真核重组表达质粒的免疫特性，将真核重组表达质粒pcDNA3.1-F和pcDNA3.1-SF肌注日本大耳白兔进行免疫，同时设置pcDNA3.1(+)、生理盐水阴性对照组和犬瘟热弱毒疫苗阳性对照组。间接ELISA法检测结果显示，免疫兔体内产生了针对CDV的特异性抗体，且抗体水平随着免疫次数和免疫剂量的增加而升高，重组质粒组体液免疫水平显著高于生理盐水组和空载体组，但不及弱毒疫苗对照组。淋巴细胞转化试验结果表明，重组质粒免疫组产生了细胞免疫，但低于弱毒疫苗组。
本研究从疑似犬瘟热病死貂肝脏中分离出1株犬瘟热病毒，并对水貂源犬瘟热病毒分离株的F基因和H基因进行了克隆与序列分析，丰富了CDV分子生物学研究内容；构建的CDV F基因真核重组表达质粒，在实验动物体内产生了特异性的体液免疫和细胞免疫。为进一步研究CDV F和H基因的免疫保护作用，研制诊断和预防水貂犬瘟热新型、高效的诊断试剂和DNA疫苗奠定了基础。
Canine distemper (CD), caused by canine distemper virus (CDV) which belongs to the Morbillivirusgenus of the Paramyxoviridae virus family, is a acute and highly contagious disease causing considerabledamages to the fur animal farming industry, threatening wildlife conservation and zoo animal. With thechanges in the environment and CDV adaptation to prevailing factors, CDV natural host's range expandsincessantly. In addition, the new CDV variants leading to the incidence and mortality rate of CD infectedanimals are increasing, and prevalent and clinical symptoms developed are more and more complicated atthe present time. Therefore, its harmful effects become more serious. In particular, when giant panda andmacaque and so forth quadrumana are infected CDV, CDV pathogenesis became protrusion. Latestresearch confirms that CDV can infect human prosoma osteoclast and that indicates CDV natural hostextends the possibility that it could infect human beings. It may be the second viral infection diseasepassing from dog to human beings.
Due to worldwide distribution of CDV and the widespread natural host and huge cross infection,eliminating CDV is almost impossible. However, a vaccine can be used as an active immunity to controlCD. Traditional attenuated vaccine has intrinsic shortcomings despite its important role in controlling theoccurrence of CD. In order to develop a new kind of safe and efficient CDV genetically engineeredvaccine, we have conducted the following works:
1. Isolation and Identification of Canine Distemper Virus from Mink
A strain of Canine Distemper Virus was isolated and identified systemically from the liver of a deadmink suspected of having Canine Distemper, at a mink farm in Qingdao, Shandong Province. The resultsindicated that typical and disciplinary CDV cell pathological changes appeared and the physical andchemical characteristics were similar to that of CDV after the virus strain had inoculated Vero cell. Thecytopathogenic effect can be interdicted by Rabbit anti-CDV positive serum. Rotundity and ellipseeosinophil inclusion body can be seen in cytoplasm during inclusion body examination. Indirectimmunofluorescent assay showed that BHK-21 cell inoculated the CDV resulting in idiosyncratic brightgreen fluorescence and that green fluorescence did not appeared in the normal cell. The total RNAextracted from Vero cell culture inoculated with the virus were amplified with Reversetranscription-polymerase chain reaction(RT-PCR), and two objective segments (324bp and 1053bp) wereobtained Thus, the virus strain was proved to be CDV and named as CDVSJ.
2. Cloning and Sequencing Analysis on hemagglutinin (H) genes of Isolated Canine Distemper Virus Strain
H gene of CDVSJ strain was amplified with RT-PCR, and the purified RT-PCR products werelinked with pMD-18T Simple Vector, then transferred to E.coli JM109 competent cell. The recombinedcloning plasmid pMD-18T-CDVH was created and sequenced. Sequencing result indicated that H genesection of CDVSJ strain was of a 1596bp fragment encoding 532 amino acids, which contained 5potential N-glycosylation positions located in 76Aa～78Aa, 318Aa～320Aa, 349Aa～351Aa, 383Aa～385Aa, 514Aa～516Aa; and also contained 12 cysteine residues located at 66, 81, 115, 211,223, 304,309, 317, 417, 493, 502, 529 positions. Compared with the nucleotide sequences of 01-2689, 007Lm,2544Han95, 25259, A75/17, American dog, black leopard, Liud, CDTaichung, Convac, Danish dog,Danish mink, DK91D,Greenlandic dog, Hamatsu, Javelina, KDK-1, LP, Onderstepoort, Snyder Hill,Tanu96, TN, Ueno and Yanaka strains, the homology is 90.7%, 91.0%, 91.9%, 92.2%, 92.0%, 91.2%,91.8%, 91.9%, 90.9%, 96.9%, 92.0%, 92.4%, 92.0%, 92.1%, 90.9%, 91.8%, 91.1%, 92.5%, 96.6%,98.6%, 91.1%, 91.1%, 91.0% and 91.0%, respectively. Derivative amino acids homology are 89.9%,90.1%, 91.4%, 90.2%, 91.9%, 91.4%, 91.9%, 91.7%, 91.2%, 95.1%, 91.4%, 91.9%, 91.6%, 91.4%,90.4%, 91.2%, 90.8%, 91.4%, 94.6%, 97.0%, 90.8%, 90.6%, 91.0% and 90.8%, respectively. Comparedwith Onderstepoort, Convac, Hamatsu, A75/17 and TN strains, CDVSJ strain contained 5 potentialN-glycosylation positions according to derivative amino acids sequence analysis. There were two moreN-glycosylation position at 318Aa～320Aaand383Aa～385Aa compared with the Onderstepoort vaccinestrain. One N-glycosylation positions, 530Aa～532Aa, were lacking compared with Convac and A75/17strains. Three N-glycosylation positions, 236Aa～238Aa, 511Aa～513Aa and 530Aa～532Aa, werelacking compared with Hamatsu strains.Two N-glycosylation position, 236Aa～238Aa and 511Aa～513Aa was lacking compared with TN strain. Clustering analysis results showed that CDVSJ strain isclosely related to the Snyder Hill strain. Sequence homologies are a close comparison to Onderstepoortand Convac, which form one group. However, the kinship between them and standard virulent strainHamatsu is far away.
3. Cloning and Sequence Analysis of fusion (F) Gene of Canine Distemper Virus Strain
F gene of CDVSJ strain was amplified with RT-PCR and the purified RT-PCR product was linkedto pMD-18T Simple Vector, then transferred to E.coli JM109 competent cell. The recombined cloningplasmid pMD-18T-CDVF was built and sequenced. Sequencing results indicated that F gene section ofCDVSJ strain was of a 1053bp fragment encoding 351 amino acids of which 9 were cysteine residues.Compared with the nucleotide sequences of 01-2601, 2544-Han95, 5804P, 25259, A75-17, DOGDK91C,ONP, PDV-2 and 98-2646strains, homology are 95.4%, 93.8%, 93.9%, 94.3%, 94.5%, 93.9%, 97.3%,94.7% and 94.1%, respectively. Derivative amino acids homology are 97.4%, 96.6%, 97.7%, 98.0%,97.4%, 97.4%, 97.2%, 98.0% and 96.6%, respectively. Antigen index comparative results showed that there are great differences in 40-50th amino acids between isolated CDVSJ and vaccine Onderstepoortand the virulent A75-17 strain.Compared with Onderstepoort, A75/17 and PDV-2 strains, the number andposition of cysteine residues of F protein in each strain was the same, being 9. That determines that CDVF gene is a comparative conservative structural protein gene.
4. Eukaryotic expression of Canine Distemper Virus F gene
Cloned F gene segment in pMD-18T simple vector was re-cloned to pcDNA3.1 (+) eukaryonexpression vector plasmid, forming eukaryon recombinant expression plasmid pcDNA3.1-CDVF andpcDNA3.1-CDVSF successfully. Eukaryon recombinant expression plasmid pcDNA3.1-CDVF andpcDNA3.1-CDVSF were transfected into BHK-21 cell by using lipoplast mediate method and has beenproved with ELISA, indirect fluorescent antibody technique and RT-PCR detection that the objectivegene was successfully expressed in mammalian cell.
5. Immunization test of Eukaryon recombinant expression plasmid
In order to study immunity characteristics of Eukaryon recombinant expression plasmid, Eukaryonrecombinant expression plasmid pcDNA3.1-F and pcDNA3.1-SF were injected intramuscularly intoJapanese albino rabbit. Meanwhile, pcDNA3.1 (+), normal saline negative control and CDV vaccinepositive were used as control. ELISA results showed that specificity antibody to CDV was produced inthe Japanese albino rabbit. Moreover antibody level increased with increasing immunizing times anddoses. The humoral immunity level of recombinant plasmid group was significantly higher than those ofnormal saline and pcDNA3.1 (+) control groups, however, but lower than that of attenuated vaccinegroup. Lymphocyte transformation test results showed that recombinant plasmid immunity groupproduced cellular immunity, but was lower than that of the attenuated vaccine group.
A strain of CDV was isolated from the liver of the suspect CD dead mink, and H and F gene ofCDVSJ strain were cloned and sequenced, providing useful information for molecular biological study ofthe virus. The recombined eukaryotic expression plasmid of CDV F protein gene was successfullyexpressed in the body of laboratory animal, producing both humoral and cell immunity. The results of thestudy provide fundamental information for the further study on the protective function of CDV H and Fgene, for the development of new type and efficient diagnostic reagent for diagnosis and prevention ofCD and DNA vaccine.
引文
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