WO2020258757A1 - 一种突变株3型鸭甲肝病毒ch-p60-117c株及构建方法 - Google Patents

一种突变株3型鸭甲肝病毒ch-p60-117c株及构建方法 Download PDF

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WO2020258757A1
WO2020258757A1 PCT/CN2019/125293 CN2019125293W WO2020258757A1 WO 2020258757 A1 WO2020258757 A1 WO 2020258757A1 CN 2019125293 W CN2019125293 W CN 2019125293W WO 2020258757 A1 WO2020258757 A1 WO 2020258757A1
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virus
strain
duck
mutant strain
duck hepatitis
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程安春
文兴建
汪铭书
吴莉萍
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四川农业大学
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    • C12N2770/00011—Details
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    • C12N2770/32451—Methods of production or purification of viral material
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Definitions

  • the invention belongs to the technical field of genetic engineering, and specifically relates to a mutant strain 3 duck hepatitis A virus CH-P60-117C strain and a construction method thereof.
  • Duck viral hepatitis (Duck viral hepatitis, DVH) is an acute and highly contagious infectious disease caused by duck hepatitis virus (DHV) infecting ducklings.
  • DDV duck hepatitis virus
  • the disease mainly affects ducklings within four weeks of age. It has the characteristics of rapid onset, rapid spread, short course of disease and high mortality.
  • the clinical manifestation is that the ducklings have spasms before death, their heads and backs are tilted back, showing "angular arch reflex".
  • the pathological changes are mainly the liver enlargement, inflammation and a large number of hemorrhagic spots.
  • the disease is mainly caused by Duck hepatitis A virus (DHAV) belonging to the genus Avian Hepatitis of the Picornavirus family.
  • DHAV has three serotypes, namely type 1, type 2 and type 3.
  • the prevalent DHAV in my country is mainly duck hepatitis A virus type 1 (DHAV-1) and duck hepatitis A virus type 3 (DHAV-3).
  • Reverse genetic manipulation technology is an important platform for carrying out viral molecular biology research. It can perform manual operations such as gene knockout and site-directed mutagenesis on RNA virus genomes in vitro, and can play an important role in elucidating the pathogenic mechanism of viruses and vaccine development. It has the advantage of being shorter than natural mutagenesis cycle.
  • the key to traditional RNA virus infectious cloning methods is to obtain a full-length cDNA clone of the viral genome, and the viral genome needs to be cloned into a suitable vector after being converted into cDNA.
  • the full-length cDNA of some viruses cannot be cloned or although they can be cloned into the vector, they are prone to mutation in the host bacteria, resulting in failure to successfully rescue the virus.
  • a technology called "Infectious Subgenomic Amplicons” has been confirmed to be able to achieve artificial rescue of single-stranded positive-stranded RNA viruses in mammalian or mosquito cells, and has been applied in Japan Encephalitis virus, West Nile virus, Zika virus, yellow fever virus, dengue virus and human Coxsackie virus and other viruses in reverse genetic research. This technology is a new type of "bacterial-free" reverse genetics method.
  • infectious subgenomic replicons first, by PCR, overlapping non-infectious subgenomic DNA fragments containing the entire viral genome are generated, and the number of overlapping subgenomic replicons can be 3 to 10, with an overlapping region of about 100 bp between adjacent replicons. At the same time, the 5'end of the first fragment and the 3'end of the last fragment are flanked by the early promoter of cytomegalovirus (Cytomegalovirus immediate early promoter).
  • PCMV hepatitis delta virus ribozyme
  • HDVR hepatitis delta virus ribozyme
  • SV40pA monkey vacuolar virus early mRNA polyadenylation signal
  • DHAV-1 attenuated vaccine mainly the use of DHAV-1 attenuated vaccine, and there is still a lack of efficient DHAV-3 live vaccine.
  • DHAV host tropism and toxicity Basic research on virus genes with variable virulence and key sites can provide a theoretical basis for the prevention and treatment of duck hepatitis. It is also urgent to obtain virus strains with variable host tropism and virulence.
  • the technical problem to be solved by the present invention is to obtain a candidate strain of duck hepatitis A virus vaccine type 3 by genetic modification; at the same time, obtaining a virus strain with changes in host tropism and virulence can be a virus gene for studying changes in DHAV host tropism and virulence And key sites, etc. to provide basic materials.
  • a mutant strain 3 duck hepatitis A virus CH-P60-117C strain (DHAV-3 CH-P60-117C), the mutant strain is based on the 117th nucleus of the 5'UTR of the virulent type 3 duck hepatitis A virus genome
  • the nucleotide acid was mutated from A to C; the 1142th nucleotide was mutated from T to A, so that the 164th amino acid of the VPO protein of the virus was mutated from the parent strain's tyrosine to asparagine; the 4334th nucleotide was changed from C was mutated to A, so that the amino acid at position 71 of the virus 2C protein was mutated from the leucine of the parent strain to isoleucine.
  • the mutant CH-P60-117C was deposited in the China Type Culture Collection on December 2, 2018, and the deposit number is CCTCC NO: V201860.
  • the virulent strain of duck hepatitis A virus type 3 is deposited in the China Type Culture Collection, and the deposit number is CCTCC NO: V201305.
  • the G mutation at the 3403th nucleotide in the genome of the mutant strain is T, as a genetic marker for infectious clones, the mutant strain can be distinguished from the parent strain and the wild strain by the PCR method combined with DNA sequencing.
  • the mutant strain containing genetic markers of the present invention can stably propagate and pass on 9-day-old duck embryos with higher virus titer, and can also stably propagate and pass on 9-day-old chicken embryos with good genetic stability. , No mutations appeared after 10 consecutive passages.
  • the mutant strain containing genetic markers of the present invention has significantly reduced pathogenicity to ducklings, and the mutant strain successfully replicates in ducklings but is not pathogenic to ducklings.
  • the mutant strain can also be used in basic research such as viral genes and key sites that change the host tropism and virulence of duck hepatitis virus.
  • a method for constructing the above mutant strain which includes the following steps:
  • the parent virus's full gene composition was divided into three fragments of similar size (2.6kb, 2.6kb and 2.7kb) for PCR amplification, and the first fragment of the virus genome was added to the 5'end of the cytomegalovirus early promoter (Cytomegalovirus). immediate early promoter, pCMV) and introduced mutation sites in the 5'UTR and VP0 genes respectively, introduced mutation sites in the 2C gene of the second fragment, and nonsense mutations in the 2A gene as genetic marker sites.
  • pCMV immediate early promoter
  • pCMV immediate early promoter
  • introduced mutation sites in the 5'UTR and VP0 genes respectively, introduced mutation sites in the 2C gene of the second fragment, and nonsense mutations in the 2A gene as genetic marker sites.
  • the DNA fragment constitutes the "infectious subgenomic replicon" of the type 3 duck hepatitis A virus mutant strain CH-P60-117C;
  • first fragment and the third fragment contain 74 and 83 base pairs of overlapping regions with the second fragment, respectively.
  • Using the method of the present invention to obtain a type 3 duck hepatitis A virus mutant strain requires a shorter period than natural mutagenesis and traditional reverse genetic manipulation techniques, and can speed up the cultivation of type 3 duck hepatitis A virus attenuated vaccine strain and virus pathogenicity The process of mechanism research.
  • the mutant strain CH-P60-117C obtained in the invention has similar antigenicity to its parent strain and can maintain stable genetic characteristics during continuous passage. Therefore, the mutant strain can be used as a candidate vaccine virus for type 3 duck hepatitis A virus. At the same time, it has higher proliferation efficiency and virus titer in duck embryos than the parent strain, can multiply in chicken embryos, and has a wider source of materials for culturing viruses. If used in vaccine production, it can increase yield and reduce costs.
  • the mutant CH-P60-117C obtained in the present invention has similar antigenicity to its parent strain. Therefore, the mutant strain can be used as a candidate vaccine strain for type 3 duck hepatitis A virus immunity, and can be used as a production strain Prepare 3 duck hepatitis A virus vaccine.
  • the mutant strain CH-P60-117C obtained in the present invention replicates in ducklings but is not pathogenic to ducklings, has good safety, and can be used as a candidate strain for attenuated vaccine of type 3 duck hepatitis A virus.
  • the mutant strain CH-P60-117C obtained in the present invention has reduced virulence and can also multiply in chicken embryos, so it can be used for duck hepatitis virus host tropism and virulence changes in viral genes and Key sites and other basic research applications.
  • Figure 1 is a schematic diagram of constructing a molecular marker mutant CH-P60-117C "infectious subgenomic replicon" based on type 3 duck hepatitis A virus as the backbone;
  • Figure 2 shows the result of transfection of duck embryo fibroblasts with the type 3 duck hepatitis A virus mutant CH-P60-117C "infectious subgenomic replicon";
  • Figure 3 shows the sequencing results of the 3403th nucleotide molecular genetic marker site of the mutant CH-P60-117C virus genome
  • Figure 4 shows the nucleotide sequencing results of the 117th nucleotide target mutation site of the mutant CH-P60-117C virus genome
  • Figure 5 is the nucleotide sequencing result of the target mutation site of nucleotide 1142 in the genome of the mutant CH-P60-117C virus;
  • Figure 6 shows the nucleotide sequencing results of the target mutation site at nucleotide 4334 of the mutant CH-P60-117C virus genome.
  • Duck hepatitis A virus type 3 virulent strain isolated in our laboratory and deposited in the Chinese Type Culture Collection of Wuhan University, China. The deposit number is: CCTCC NO: V201305, classification and designation: Avian hepatitis is a Picornaviridae 3 Type Duck Hepatitis A Virus type 3, DHAV-3).
  • TaKaRa MiniBEST Universal RNA Extraction Kit PrimeSTAR Max DNA Polymerase, DNA Marker, etc. were purchased from Bao Bioengineering (Dalian) Co., Ltd.; gel recovery kits, plasmid extraction kits, etc. were purchased from Omega, USA; liposome transfection kits Lipofectamine 3000 was purchased from Invitrogen; other reagents are all domestically pure.
  • Example 1 Construction and virus rescue of type 3 duck hepatitis A virus mutant strain CH-P60-117C "infectious subgenomic replicon"
  • RNA Extraction kit extract the whole genome RNA of the type 3 duck hepatitis A virus isolate from duck embryo allantoic fluid, and use a nucleic acid protein detector (Bio Rad, Smartspec3000) to determine its After nucleic acid concentration and purity, store at -70°C for later use.
  • DHAV-3-F1-A117C, DHAV-3-F1-A117C-T1142A and DHAV-3-F1-T1142A fragments are first fused to F1-A117C-T1142A, Then fuse pCMV and F1-A117C-T1142A fragments into pCMV-F1; fuse DHAV-3-F2-1 fragments, DHAV-3-F2-C4334A-1 fragments and DHAV-3-F2-C4334A-2 fragments into F2 Fragment:
  • the DHAV-3-F3-HDVR and HDVR-SV40pA fragments are fused to the F3-HdvRz/SV40pA fragment, and the three DNA fragments finally obtained constitute the mutant CH-P60-117C "infectious subgenomic replicon".
  • the gel recovery kit (Omega) was used to cut the gel to recover the target fragments.
  • the DNA fragments were sent to Shanghai Shenggong Biological Engineering Co., Ltd. for sequencing.
  • reverse genetics was used to change the 3403 base of the mutant strain’s genome from the mutation G to T, and the mutation does not change 2A.
  • the composition of the corresponding amino acid of the protein which is used as a molecular genetic marker site, can be used to distinguish mutant strains from parental strains and wild virus strains by PCR combined with DNA sequencing.
  • the rescued virus was purified 5 times by limiting dilution on duck embryos. Total RNA is extracted from the allantoic fluid. After reverse transcription, the DNA fragments containing the mutation sites are amplified by PCR using F2-F and F2-R primers.
  • the amplified fragments are separated by 1% agarose gel electrophoresis, and then the gel is used
  • the recovery kit (Omega) was cut and recovered, and the DNA fragments were sent to Shanghai Shenggong Bioengineering Co., Ltd. for sequencing.
  • the sequencing results showed that the amplified product contained the introduced silent mutation (G3403T), as shown in Figure 3, indicating that we Get the correct rescue virus, not the parental strain or the wild-type strain.
  • the rescue virus of the first generation was diluted 1:100 with sterile normal saline and inoculated with 5 9-day-old duck embryos or chickens. Embryo. The results showed that the death time of duck embryos/chicken embryos was concentrated between 24 hours and 48 hours after inoculation. The embryos had obvious lesions and were successively passaged 10 times. During this period, the virus liquid of each generation was collected and stored in the refrigerator at -80°C.
  • the ELD50 is calculated according to the Reed-Muech method.
  • the virus content in each 0.2mL allantoic fluid is 10 -7.55 ELD50 and 10 -4.50 ELD50, mutant strain duck embryo proliferation was significantly stronger than the parent strain, such as an antigen for vaccine production can increase yield and reduce costs.
  • the mutant strain of duck hepatitis A virus type 3 CH-P60-117C could multiply in the chicken embryo and cause the death of the chicken embryo.
  • the virus titer of the mutant strain in the chicken embryo allantoic fluid It is 10 -6.55 ELD50; while the parent strain cannot proliferate and lethal chicken embryos. If the mutant strain is used for antigen production in vaccines, the source of materials for culturing the virus is wider and the cost is reduced; and the host tropism of the mutant strain has changed, the obtained mutant virus can be used as the host tropism and virulence of the duck hepatitis virus. Basic materials for research on virus genes and their key sites.
  • the neutralization titer of the rabbit anti-type 3 duck hepatitis A virus serum against the mutant strain and the parent strain was determined by the fixed virus dilution serum method.
  • the rabbit anti-type 3 duck hepatitis A virus standard serum (titer ⁇ 1:128) Use sterile normal saline to make a 10-fold dilution of 9 dilutions from 2 -1 to 2 -9 , and dilute the virus to each unit dose (0.2mL) containing 200ELD50, and then the two are equal Mix and add 5% penicillin and streptomycin in a 37°C water bath for 1 hour, and then inoculate the allantoic cavity of 9-day-old healthy duck embryos at 0.2 mL per embryo, and inoculate 5 duck embryos at each dilution.
  • the duck embryos of the negative serum control group healthy rabbit serum mixed with the virus
  • blank control group sterilizized normal saline mixed with the virus
  • the duck embryos of the mutant strain and the parent strain neutralization group are all viable.
  • the dilution is 2 -7
  • the duck embryos begin to lose protection. Initially, with the higher the dilution, the lower the duck embryo protection rate, until the duck embryo loses its protection when the dilution is 2-9 . It shows that the preparation of vaccine antigens with mutant strains can also protect type 3 duck hepatitis A virus infection.
  • the parent strain and the mutant strain were used for safety tests.
  • the liver tissue of dead duck embryos in Example 2.3 was collected for homogenization treatment, and sterilized phosphate buffer solution was added at a volume ratio of 1:100, ground, and repeated freezing and thawing 3 times. After centrifugation at 12000g for 10min, the supernatant was filtered and sterilized with a 0.22 ⁇ m filter, and then inoculated with 9-day-old duck embryos through the allantoic cavity to determine the ELD50, and then the virus solution was diluted to 10 3.0 ELD50/0.4mL. In addition, 30 1-day-old healthy ducklings were randomly divided into 3 groups.
  • the ducklings in the experimental group were inoculated with 0.4mL parent strain or mutant strain by intramuscular injection, and the ducklings in the control group were inoculated with equal volume of sterilized physiological saline. Animals are kept in isolation in the animal house, free drinking water and food, daily observation after vaccination, record the morbidity and death of ducklings, timely necropsy of dead ducklings, necropsy for surviving ducklings 7 days later, record duckling liver, kidney and other organs The disease situation.
  • the ducklings in the control group showed no clinical symptoms.
  • the eating and drinking conditions of the ducklings were normal.
  • the duckling disease rate was 80%, and the mortality rate was 60%.
  • the pathogenicity of the mutant strain to the ducklings was significantly reduced.
  • the ducklings of the mutant strain have no clinical symptoms, eating and drinking conditions are normal, and the virus is detected in the cloacal swab, indicating that the mutant strain successfully replicated in the ducklings but is not pathogenic to the ducklings, and has a candidate strain as an attenuated vaccine The potential; and can distinguish mutant strains from parental strains by PCR combined with DNA sequencing.
  • the mutant strain determined according to Example 2 has higher proliferation efficiency and virus titer on duck embryos than the parent strain; it can also proliferate on chicken embryos; it has good immunogenicity and genetic stability; it is pathogenic to ducklings
  • the mutant strain can replicate in ducklings but is not pathogenic to ducklings. It shows that the mutant strain ISA-A117C-T1142A-C4334A is an ideal vaccine candidate strain and can be used to prepare duck hepatitis A virus type 3. vaccine.
  • the inactivated vaccine was tested for sterility and mycoplasma in accordance with the appendix of the current "People's Republic of China Veterinary Pharmacopoeia", and the test results were all negative.
  • the inactivated vaccine was tested for foreign viruses in accordance with the appendix of the current "People's Republic of China Veterinary Pharmacopoeia", and the test results were all negative.
  • Twenty 1-day-old ducklings were randomly divided into 2 groups. All 10 ducklings in the vaccination group were vaccinated with a dose of the vaccine prepared above by intramuscular injection in the legs, and the other group was injected with the same amount of sterilized saline as In contrast, the two groups of ducklings were reared in isolation, free to drink and eat. After inoculation, the ducklings were observed and recorded daily. After 7 days, the parent strain was challenged with 10 times the LD50 dose. After the challenge, the incidence of the ducklings was observed and recorded daily. In case of death, the dead ducklings should be necropsied in time, and the observation should be continued for 1 week. The surviving ducklings should be necropsied and the lesions of liver, kidney and other organs should be recorded.
  • the ducklings of the experimental group and the negative control group had no clinical symptoms, and the eating and drinking conditions of the ducklings were normal.
  • the ducklings in the negative control group were sluggish and had neurological symptoms.
  • 6 died.
  • the surviving ducklings had liver hemorrhage and other diseases of varying degrees.
  • the incidence rate was 80% (8/10)
  • the mortality rate was 60% (6/10), while the ducklings in the immunized group had no disease and death. They ate normal drinking water, indicating that the inactivated vaccine prepared with the mutant virus was safe and effective, and could protect the homologous virulent challenge.

Abstract

提供了一种突变株3型鸭甲肝病毒CH-P60-117C株及构建方法，具体为3型鸭甲肝病毒强毒株基因组5'UTR的第117位核苷酸由A突变为C；第1142位核苷酸由T突变为A，从而使病毒的VP0蛋白第164位氨基酸由亲本株的酪氨酸突变为天冬酰胺；第4334位核苷酸由C突变为A，从而使病毒2C蛋白的第71位氨基酸由亲本株的亮氨酸突变为异亮氨酸。CH-P60-117C株免疫原性和遗传稳定性良好，对雏鸭不致病。

Description

一种突变株3型鸭甲肝病毒CH-P60-117C株及构建方法 技术领域
本发明属于基因工程技术领域，具体涉及一种突变株3型鸭甲肝病毒CH-P60-117C株及其构建方法。
背景技术
鸭病毒性肝炎(Duck viral hepatitis，DVH)是由鸭肝炎病毒(Duck hepatitis virus，DHV)感染雏鸭引起的一种急性、高度接触性传染病。目前世界上主要养鸭地区均有本病的存在，具有间歇爆发、地方流行性等特点，是危害养鸭业的主要疾病之一。该病主要侵害四周龄以内的雏鸭，具有发病急，传播迅速，病程短和死亡率高等特点；临床主要表现为雏鸭死前发生痉挛，头向背部后仰，呈“角弓反张”，病理变化主要为剖检可见肝脏肿大发炎和大量的出血性斑点。该病主要由属于小RNA病毒科禽肝病毒属的鸭甲肝病毒(Duck hepatitis A virus，DHAV)引起。DHAV具有三种血清型，即1型、2型和3型。近年来，我国流行的DHAV主要是1型鸭甲肝病毒(DHAV-1)和3型鸭甲肝病毒(DHAV-3)。
反向遗传操作技术是开展病毒分子生物学研究的一种重要平台，可以在体外对RNA病毒基因组进行基因敲除、定点诱变等人工操作，在阐明病毒致病机制和疫苗研制中能够发挥重要作用并具比自然诱变周期短的优点。传统RNA病毒感染性克隆方法的关键是获得病毒基因组cDNA全长片段克隆，并且病毒基因组转换为cDNA后需要克隆到合适的载体中，为了避免病毒序列在细菌中的不稳定问题，研究者通常采取分段克隆方法，通过小片段连接成大片段，最后将大片段通过酶切连接的方法得到全长cDNA克隆。但是该方法酶切位点选取限制较多，体外将多个大片段进行连接效率较低，另外一些复制酶基因cDNA克隆在细菌中具有不稳定性，因此获得病毒基因组全长cDNA的整个过程不仅 操作步骤麻烦，而且耗时较长，成功率不高，同时，有些病毒的全长cDNA无法克隆或是虽然能克隆进载体但在宿主菌中易发生变异而导致不能成功拯救病毒。目前，一种被称为“感染性亚基因组复制子(Infectious Subgenomic Amplicons)”的技术已被证实能够在哺乳动物或蚊子细胞中实现对单股正链RNA病毒的人工拯救，并被应用于日本脑炎病毒、西尼罗病毒、寨卡病毒、黄热病病毒、登革病毒和人科萨奇病毒等病毒的反向遗传学研究中。该技术是一种新型的“无细菌”反向遗传学方法，不需要获得病毒全长cDNA质粒以及在体外获得病毒RNA转录本，可以直接通过转染具有同源区域的DNA片段拯救病毒。具体的的来说，“感染性亚基因组复制子”采用的技术路线是：首先通过PCR方法产生包含整个病毒基因组的重叠的非感染性亚基因组DNA片段，这些重叠的亚基因组复制子数量可以为3至10个，相邻的复制子之间具有100bp左右的重叠区域，同时，第一个片段的5'和最后一个片段的3'末端分别侧接巨细胞病毒早期启动子(Cytomegalovirus immediate early promoter，pCMV)序列、丁型肝炎病毒核酶(Hepatitis delta virus ribozyme，HDVR)序列和猴空泡病毒早期mRNA多腺苷酸化信号(SV40 early mRNA polyadenylation signal，SV40pA)序列，这些元件能够帮助亚基因组复制子在被混合转染到易感细胞后开始转录，并利用宿主细胞的同源重组机制自发重组，形成具有感染性的完整病毒转录本，继而导致病毒的复制与增殖，最终获得具有感染性的拯救病毒。
当前市场上对DHAV的防控主要是使用DHAV-1弱毒疫苗，尚缺乏高效的DHAV-3活疫苗，研发适合我国DHAV-3流行情况的新型分子标记疫苗迫在眉睫；同时，DHAV宿主嗜性及毒力变化的病毒基因及关键位点等基础研究可为鸭肝炎的防治提供理论依据，这也迫切需要获得宿主嗜性及毒力变化的病毒株。
发明内容
本发明要解决的技术问题是，利用基因改造获得3型鸭甲肝病毒疫苗候选毒株；同时，获得宿主嗜性和毒力变化的病毒株可为研究DHAV宿主嗜性和毒力变化的病毒基因及关键位点等提供基础材料。
为了实现上述技术目的，本发明具体通过以下技术方案实现：
一种突变株3型鸭甲肝病毒CH-P60-117C株(DHAV-3 CH-P60-117C)，所述的突变株为以3型鸭甲肝病毒强毒株基因组5'UTR的第117位核苷酸由A突变为C；第1142位核苷酸由T突变为A，从而使病毒的VP0蛋白第164位氨基酸由亲本株的酪氨酸突变为天冬酰胺；第4334位核苷酸由C突变为A，从而使病毒2C蛋白的第71位氨基酸由亲本株的亮氨酸突变为异亮氨酸。
所述的突变株CH-P60-117C于2018年12月2日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：V201860。
所述的3型鸭甲肝病毒强毒株保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：V201305。
所述的突变株基因组第3403位核苷酸的G突变为T，作为感染性克隆的遗传标记，可通过PCR方法结合DNA测序来区分突变株与亲本株以及野毒株。
本发明含有遗传标记的突变株与亲本病毒一样能够在9日龄鸭胚上稳定增殖传代且病毒滴度更高，还能在鸡胚9日龄鸡胚上稳定增殖传代，且遗传稳定性良好，连续传代10次未出现突变。
本发明含有遗传标记的突变株相对于亲本病毒，对雏鸭致病力已明显下降，突变株成功在雏鸭体内复制但对雏鸭不致病。
在本发明的另一方面，提供了上述突变株在制备3型鸭肝炎病毒疫苗中的应用。
同时该突变株也可用在鸭肝炎病毒宿主嗜性和毒力变化的病毒基因及关键 位点等基础研究中。
在本发明的另一方面，提供了上述突变株的构建方法，包括以下步骤：
1)将亲本病毒全基因组分为大小相近的(2.6kb、2.6kb和2.7kb)三个片段进行PCR扩增，在病毒基因组第一个片段的5'端添加巨细胞病毒早期启动子(Cytomegalovirus immediate early promoter，pCMV)且分别在5'UTR和VP0基因中引入突变位点，在第二个片段的2C基因中引入突变位点且2A基因无义突变作为遗传标记位点，在病毒基因组第三个片段的3'端添加丁型肝炎病毒核酶(Hepatitis delta virus ribozyme，HDVR)序列和猴空泡病毒早期mRNA多腺苷酸化信号(SV40 early mRNA polyadenylation signal，SV40pA)序列，最后获得三个DNA片段构成3型鸭甲肝病毒突变株CH-P60-117C的“感染性亚基因组复制子”；
(2)将构建的3型鸭甲肝病毒突变株CH-P60-117C的“感染性亚基因组复制子”与转染试剂混合后转染鸭胚成纤维细胞，复制子在宿主细胞内转录并利用细胞的同源重组机制自发重组，形成具有感染性的完整病毒转录本，继而导致病毒的复制与增殖，最终获得含有遗传标记的3型鸭甲肝病毒突变株CH-P60-117C。
进一步的，第一个片段和第三个片段分别与第二个片段含有74个和83个碱基对的重叠区域。
本发明的有益效果为：
1、利用本发明方法获得3型鸭甲肝病毒突变株比自然方法诱变和传统反向遗传操作技术的所需周期更短，可加快3型鸭甲肝病毒弱毒疫苗毒株的培育和病毒致病机制研究的进程。
2、发明中获得的突变株CH-P60-117C具有与其亲本毒株相似的抗原性且在 连续传代过程中能够保持稳定的遗传特性，因此该突变株可作为3型鸭甲肝病毒免疫候选疫苗毒株；同时其在鸭胚中具有比亲本株更高的增殖效率和病毒滴度、能够在鸡胚中增殖，培养病毒的材料来源更广。如用于疫苗生产中，可提高产量、降低成本。
3、本发明中获得的突变株CH-P60-117C具有与其亲本毒株相似的抗原性，因此该突变株可作为3型鸭甲肝病毒免疫候选疫苗毒株，可使用其作为生产毒株用于制备3型鸭甲肝病毒疫苗。
4、本发明中得到的突变株CH-P60-117C在雏鸭体内复制但对雏鸭不致病，具有良好的安全性，可作为3型鸭甲肝病毒弱毒疫苗候选毒株。
5、由于本发明中获得的突变株CH-P60-117C和亲本株相比，毒力降低、还能够在鸡胚中增殖，因此可用于鸭肝炎病毒宿主嗜性和毒力变化的病毒基因及关键位点等基础研究的应用中。
附图说明
图1为基于3型鸭甲肝病毒为骨架构建分子标记突变株CH-P60-117C“感染性亚基因组复制子”的示意图；
图2为3型鸭甲肝病毒突变株CH-P60-117C“感染性亚基因组复制子”转染鸭胚成纤维细胞结果图；
图3为突变株CH-P60-117C病毒基因组的第3403核苷酸分子遗传标记位点测序结果；
图4为突变株CH-P60-117C病毒基因组的第117核苷酸目标突变位点核苷酸测序结果；
图5为突变株CH-P60-117C病毒基因组的第1142核苷酸目标突变位点核苷酸测序结果；
图6为突变株CH-P60-117C病毒基因组的第4334核苷酸目标突变位点核苷酸测序结果。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均可从商业途径得到。
下述实施例中所用的材料和试剂具体如下：
病毒株：
3型鸭甲肝病毒强毒株：本实验室分离得到，已保藏于中国武汉大学的中国典型培养物保藏中心，保藏编号为：CCTCC NO：V201305，分类命名：禽肝病毒属小RNA病毒科3型鸭甲肝病毒(Duck Hepatitis A Virus type 3，DHAV-3)。
试剂及仪器：
TaKaRa MiniBEST Universal RNA Extraction Kit，PrimeSTAR Max DNA Polymerase，DNA Marker等购自宝生物工程(大连)有限公司；胶回收试剂盒，质粒抽提试剂盒等购自美国Omega公司；脂质体转染试剂盒Lipofectamine 3000购自Invitrogen公司；其他试剂均为国产分析纯。
核酸蛋白检测仪(Bio Rad，Smartspec 3000)、梯度PCR仪(Biometra，Tgradient)、电泳仪(Bio Rad，Powerpac 300)和凝胶成像系统(Bio Rad Versa Doc Model 2000)。
实施例1 3型鸭甲肝病毒突变株CH-P60-117C“感染性亚基因组复制子”的构建及病毒拯救
1.1.引物的设计与合成
根据GenBank中3型鸭甲肝病毒的全基因组序列，设计了9对引物用于扩 增病毒全基因组序列、pCMV和SV40pA序列，具体序列信息见表1，引物由上海生工生物工程有限公司合成。
表1 构建3型鸭甲肝病毒突变株CH-P60-117C感染性亚基因组复制子引物
Figure PCTCN2019125293-appb-000001
1.2.病毒抽提
参照TaKaRa MiniBEST Universal RNA Extraction试剂盒的使用说明书操作，从鸭胚尿囊液中抽提3型鸭甲肝病毒分离株的病毒全基因组RNA，并使用核酸蛋白检测仪(Bio Rad，Smartspec3000)测定其的核酸浓度和纯度后，-70℃保存备用。
1.3.基因片段扩增克隆
(1)使用PrimeScript II 1st Strand cDNA Synthesis试剂盒将抽提的总RNA反转录为cDNA模板，然后使用DNA高保真PCR酶PrimeSTAR Max DNA Polymerase，使用引物F1-F和A117C-R、A117C-F和T1142A-R、T1142A-F和F1-R以亲本株病毒总RNA的反转录产物为模板扩增得到DHAV-3-F1-A117C、DHAV-3-F1-A117C-T1142A和DHAV-3-F1-T1142A片段，使用引物F2-1-F和F2-1-R、F2-2-F和C4334A-R、C4334A-F和F2-2-R以亲本株病毒总RNA的反转录产物为模板分别扩增得到DHAV-3-F2-1片段、DHAV-3-F2-C4334A-1片段和DHAV-3-F2-C4334A-2片段，使用引物F3-HDVR-F和F3-HDVR-R以亲本株病毒总RNA的反转录产物为模板扩增得到DHAV-3-F3-HDVR片段；以真核表达质粒pEGFP-C1为模板，使用引物pCMV-F和pCMV-R扩增得到pCMV片段，使用引物HDVR-SV40pA-F和HDVR-SV40pA-R扩增得到HDVR-SV40pA片段。
(2)通过融合PCR技术，如图1所示，先将DHAV-3-F1-A117C、DHAV-3-F1-A117C-T1142A和DHAV-3-F1-T1142A片段融合为F1-A117C-T1142A，然后将pCMV和F1-A117C-T1142A片段融合为pCMV-F1；将DHAV-3-F2-1片段、DHAV-3-F2-C4334A-1片段和DHAV-3-F2-C4334A-2片段融合为F2片段；将DHAV-3-F3-HDVR和HDVR-SV40pA片段融合为F3-HdvRz/SV40pA片段，最终获得的三个DNA片段构成突变株CH-P60-117C“感染性亚基因组复制子”。扩增片段经1％琼脂糖凝胶电泳分离后，分别采用凝胶回收试剂盒(Omega)切胶回收目的片段。将DNA片段送至上海生工生物工程有限公司进行测序。
1.4.突变株CH-P60-117C“感染性亚基因组复制子”的转染拯救
使用9日龄鸭胚制备制备原代鸭胚成纤维细胞，当3.5cm培养皿中的细胞 生长至90％融合时，将等量(1.5μg)的pCMV-F1、F2和F3-HdvRz-SV40pA基因片段与Lipofectamine 3000(Invitrogen)混合后转染长满90％的鸭胚成纤维细胞，对照组仅使用Lipofectamine 3000(Invitrogen)进行转染。细胞置于37℃5％CO 2培养箱中进行培养观察，16小时后更换培养基，在转染72小时后，转染组细胞出现细胞碎裂现象，而对照组细胞生长状况良好。在转染鸭胚成纤维细胞120小时后，细胞生长状况如图2所示，拍照记录后将细胞反复冻融3次，细胞培养液通过尿囊腔途径接种5枚9日龄鸭胚，0.2mL/枚，石蜡封口后放入孵化箱内继续孵育，每隔8小时照胚1次，观察鸭胚在接种后的死亡情况，弃去24小时内死亡鸭胚。结果显示鸭胚在接种后24小时至48小时之间死亡，鸭胚死亡胚体出血严重，收集尿囊液作为第一代反向遗传病毒株进行保存。
实施例2 3型鸭甲肝病毒突变株CH-P60-117C的鉴定和特性
2.1.拯救病毒中遗传标记的鉴定
为了排除拯救病毒可能来自于转染或传代过程中亲本病毒或野毒株污染的可能，利用反向遗传学方法，突变株基因组的3403位碱基由突变G突变为T，该突变不改变2A蛋白相应氨基酸的组成，该位点作为分子遗传标记位点，可通过PCR方法结合DNA测序来区分突变株与亲本株以及野毒株。拯救病毒在鸭胚上通过有限稀释法传代纯化5次。从尿囊液中提取总RNA，反转录后使用F2-F和F2-R引物PCR扩增含有突变位点的DNA片段，扩增片段经1％琼脂糖凝胶电泳分离，然后使用凝胶回收试剂盒(Omega)切胶回收，并将DNA片段送至上海生工生物工程有限公司进行测序，测序结果显示扩增的产物含有引入的沉默突变(G3403T)，如图3所示，表明我们获得了正确的拯救病毒，而非亲本株或者野生型毒株污染。
2.2.拯救病毒及其突变位点的遗传稳定性检测
为观察拯救出来的反向遗传病毒的是否能够在鸭胚和鸡胚中增殖传代，将第1代的拯救病毒用灭菌生理盐水做1:100稀释，接种5枚9日龄鸭胚或鸡胚。结果显示鸭胚/鸡胚死亡时间集中在接种后24小时至48小时之间，胚体病变明显，并连续传代10次，期间收集每一代病毒液，放于-80℃冰箱保存。使用第1、5和10代病毒液提取RNA，反转录为cDNA后，PCR扩增含有突变位点的DNA片段，并对突变位点以及遗传标记位点进行检测，如图4、图5和图6所示，测序结果显示第1、5和10代毒未出现突变，表明此病毒遗传稳定性良好。
2.3.病毒增殖及含量测定
用无菌生理盐水溶液倍比稀释3型鸭甲肝病毒拯救病毒与亲本株，选择10 -3、10 -4、10 -5、10 -6、10 -7、10 -8这6个稀释度，分别以每胚0.2mL的量经尿囊腔途径接种5枚9日龄的鸭胚，另设灭菌生理盐水对照5枚，接种后置于37℃恒温培养箱内孵育，24小时内死亡鸭胚不计，观察并记录7天内接种鸭胚的死亡和存活情况，按Reed-Muech法计算ELD50，结果显示拯救病毒与亲本株病毒具有不同的增殖能力，每0.2mL尿囊液中病毒含量分别为10 -7.55ELD50和10 -4.50ELD50，突变株在鸭胚中的增殖能力明显强于亲本株，如用于疫苗中抗原生产可增加产量、降低成本。
另外，在9日龄鸡胚上进行相同实验操作，结果表明3型鸭甲肝病毒突变株CH-P60-117C能够在鸡胚中增殖并导致鸡胚死亡鸡胚尿囊液中突变株病毒滴度为10 -6.55ELD50；而亲本株不能在鸡胚中增殖和致死鸡胚。突变株如用于疫苗中抗原生产，培养病毒的材料来源更广并可降低成本；而且突变株的宿主嗜性发生了变化，获得的突变病毒可作为鸭肝炎病毒宿主嗜性和毒力变化的病毒基因及其关键位点等研究的基础材料。
2.4.血清中和试验检测病毒的抗原性
为检测反向遗传株在鸭胚传代中是否产生成熟的子代病毒粒子以及病毒抗原性是否发生改变。采用固定病毒稀释血清法测定兔抗3型鸭甲肝病毒血清对突变株与亲本株的中和效价，首先将发明人所在实验室前期制备的兔抗3型鸭甲肝病毒标准血清(效价≥1:128)用灭菌生理盐水作10倍倍比稀释从2 -1到2 -9这9个稀释度，同时将病毒稀释成每个单位剂量(0.2mL)含200ELD50，然后两者等量混合并加入5％的青、链霉素双抗37℃水浴1小时，然后以每胚0.2mL的计量接种9日龄健康鸭胚尿囊腔内，每个稀释度接种5枚鸭胚。另设阴性血清对照组(健康兔血清与病毒混合)和空白对照组(灭菌生理盐水与病毒混合)，弃去24小时内死亡的鸭胚，观察并记录7天内的鸭胚死亡和存活的情况，然后计算兔抗3型鸭甲肝病毒标准血清对病毒的中和效价。
结果表明拯救病毒和亲本株病毒具有相同的抗原性，阴性血清对照组(健康兔血清与病毒混合)和空白对照组(灭菌生理盐水与病毒混合)的鸭胚均在24小时至48小时间全部死亡；当血清稀释度在2 -1至2 -6之间时，突变株和亲本株中和组的鸭胚全部健活，当稀释度在2 -7时鸭胚开始失去保护，此时开始，随稀释度越高，鸭胚保护率越低，直到稀释度为2 -9时鸭胚彻底失去保护。表明如用突变株制备疫苗抗原也可保护3型鸭甲肝病毒的感染。
2.5.对易感雏鸭的毒力和安全性试验
使用亲本株和突变株进行安全性试验，采集实施例2.3中死亡鸭胚的肝脏组织匀浆处理，按体积比1：100的比例加入灭菌磷酸盐缓冲溶液，研磨，反复冻融3次，12000g离心10min，上清液经0.22μm滤器过滤除菌后经尿囊腔途径接种9日龄鸭胚测定其ELD50，然后将病毒液稀释至10 3.0ELD50/0.4mL。另外将1日龄健康雏鸭30只随机分为3组，试验组的雏鸭经肌肉注射途径接种0.4mL亲本株或突变株，对照组的雏鸭则接种等体积灭菌生理盐水，在不同动物房 内隔离饲养，自由饮水采食，接种后每日观察，记录雏鸭的发病、死亡情况，及时剖检死亡雏鸭，观察7天后剖检存活雏鸭，记录雏鸭肝脏、肾脏等器官的病变情况。
7天内对照组雏鸭未出现临床症状，雏鸭进食和饮水状况正常，亲本株接种后雏鸭发病率80％，死亡率为60％，而突变株对雏鸭致病力已明显下降，接种突变株的雏鸭未出现临床症状，进食和饮水状况正常，且泄殖腔拭子中检测到病毒，表明突变株成功在雏鸭体内复制但对雏鸭不致病，有作为弱毒疫苗的候选毒株的潜力；且可通过PCR方法结合DNA测序来区分突变株与亲本株。
实施例3 3型鸭甲肝病毒突变株CH-P60-117C在制备灭活疫苗中的应用及效果评价
根据实施例2中所测定的突变株在鸭胚上比亲本株具有更高的增殖效率和病毒滴度；还能够在鸡胚上增殖；免疫原性和遗传稳定性良好；对雏鸭致病力已明显下降，突变株能够在雏鸭体内复制但对雏鸭不致病等特性，表明突变株ISA-A117C-T1142A-C4334A是一株理想的疫苗候选株，可用于制备3型鸭甲肝病毒疫苗。
3.1疫苗的制备方法
将3型鸭甲肝病毒突变株CH-P60-117C作为种毒用灭菌生理盐水稀释100倍，尿囊腔接种20枚9日龄鸭胚，每胚0.2mL，置37℃恒温培养箱内继续孵育，接种后24小时后照蛋1次，弃去死胚，以后每隔8小时照蛋1次，将死亡的鸭胚随时取出，至48小时，鸭胚全部死亡，将收集的鸭胚气室向上直立，置于4℃冷却8小时，将冷却后的无菌收集鸭胚尿囊液，置于-20℃保存备用。
采用终浓度为0.1％的甲醛溶液处理病毒液，37℃灭活24小时，然后使用灭菌生理盐水将病毒含量稀释至10 3ELD50/0.1mL后与弗氏不完全佐剂等体积乳 化混匀，即制成3型鸭甲肝病毒突变株株灭活疫苗。
3.2无菌和支原体检测
将灭活疫苗按照现行《中华人民共和国兽药典》附录进行无菌和支原体检验，检测结果均为阴性。
3.3外源病毒检测
将灭活疫苗按照现行《中华人民共和国兽药典》附录进行外源病毒检测，检测结果均为阴性。
3.4疫苗的安全性试验
为检验疫苗的安全性，以10倍免疫剂量免疫10只1日龄雏鸭，每只经腿部肌肉接种途径接种0.2mL，同时取10只雏鸭注射灭菌生理盐水作为对照组，隔离饲养，自由饮水采食，每日观察记录雏鸭的健康情况，观察7天后剖解雏鸭，结果免疫组与对照组雏鸭在接种7天内均无发病，且剖解结果显示，雏鸭各免疫器官及病毒嗜性组织中均未出现病理变化，且泄殖腔拭子中未检测到病毒，表明疫苗对1日龄雏鸭没有致病性。
3.5疫苗的免疫效力
1日龄雏鸭20只随机分为2组，疫苗接种组的10只雏鸭均经腿部肌肉注射途径接种一羽份上述制备得到的疫苗免疫，另一组注射等量灭菌生理盐水作为对照，两组雏鸭隔离饲养，自由饮水采食，接种后每日观察记录雏鸭的情况，7天后用10倍LD50剂量亲本株进行攻毒，攻毒后每日观察记录雏鸭的发病、死亡情况，及时剖检死亡雏鸭，持续观察1周，剖检存活雏鸭，记录肝脏、肾脏等器官的病变情况。
免疫后7天内实验组和阴性对照组雏鸭均未出现临床症状，雏鸭进食和饮水状况正常。攻毒后第2天阴性对照组有雏鸭精神不振，出现神经症状，至第7 天，共6只死亡，存活雏鸭有不同程度的肝脏出血等病变，发病率80％(8/10)，死亡率60％(6/10)，而免疫组雏鸭无发病死亡，正常饮水采食，表明用突变株病毒制备的灭活疫苗安全有效，可以保护同源强毒的攻毒。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种突变株3型鸭甲肝病毒CH-P60-117C株(DHAV-3 CH-P60-117C)，其特征在于，所述的突变株3型鸭甲肝病毒CH-P60-117C于2018年12月2日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：V201860。
  2. 根据权利要求1所述的一种突变株3型鸭甲肝病毒CH-P60-117C株，其特征在于，所述的突变株为以3型鸭甲肝病毒强毒株基因组5'UTR的第117位核苷酸由A突变为C；第1142位核苷酸由T突变为A，从而使病毒的VP0蛋白第164位氨基酸由亲本株的酪氨酸突变为天冬酰胺；第4334位核苷酸由C突变为A，从而使病毒2C蛋白的第71位氨基酸由亲本株的亮氨酸突变为异亮氨酸。
  3. 根据权利要求1和权利要求2所述的一种突变株3型鸭甲肝病毒CH-P60-117C株，其特征在于，所述的3型鸭甲肝病毒强毒株保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：V201305。
  4. 根据权利要求1和权利要求2所述的一种突变株3型鸭甲肝病毒CH-P60-117C株，其特征在于，所述的突变株基因组第3403位核苷酸的G突变为T，作为感染性克隆的遗传标记。
  5. 权利要求1和权利要求2所述的突变株3型鸭甲肝病毒CH-P60-117C的构建方法，其特征在于，包括以下步骤：
    1)将亲本病毒全基因组分为大小相近的三个片段进行PCR扩增，在病毒基因组第一个片段的5'端添加巨细胞病毒早期启动子且分别在5'UTR和VP0基因中引入突变位点，在第二个片段的2C基因中引入突变位点，在病毒基因组第三个片段的3'端添加丁型肝炎病毒核酶序列和猴空泡病毒早期mRNA多腺苷酸化信号序列，获得三个DNA片段构成突变株DHAV-3 CH-P60-117C的感染性亚基因组复制子；
    2)将构建的突变株DHAV-3 CH-P60-117C的感染性亚基因组复制子与转染试剂混合后转染鸭胚成纤维细胞，复制子在宿主细胞内转录并利用细胞的同源重组机制自发重组，形成具有感染性的完整病毒转录本，继而导致病毒的复制与增殖，最终获得含有遗传标记的突变株DHAV-3 CH-P60-117C。
  6. 根据权利要求5所述的构建方法，其特征在于，第一个片段和第三个片段分别与第二个片段含有74个和83个碱基对的重叠区域。
  7. 根据权利要求5所述的构建方法，其特征在于，在第二个片段的2A基因中引入无义突变作为遗传标记位点。
  8. 权利要求1和权利要求2所述的突变株3型鸭甲肝病毒CH-P60-117C株在制备3型鸭肝炎病毒疫苗中的应用。
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