WO2018000708A1 - 一种番鸭细小病毒亚单位疫苗 - Google Patents

一种番鸭细小病毒亚单位疫苗 Download PDF

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WO2018000708A1
WO2018000708A1 PCT/CN2016/107376 CN2016107376W WO2018000708A1 WO 2018000708 A1 WO2018000708 A1 WO 2018000708A1 CN 2016107376 W CN2016107376 W CN 2016107376W WO 2018000708 A1 WO2018000708 A1 WO 2018000708A1
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protein
muscovy duck
muscovy
duck parvovirus
vaccine
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类延乐
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类延乐
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • C12N2750/14034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of poultry vaccine preparation, and particularly relates to a muscovy duck parvovirus subunit inactivated vaccine.
  • Parvovirus disease is a highly contagious disease of geese and muscovy ducks (also known as swallow ducks). It is also known as Debussy's disease, goose flu, goose or gosling, goose hepatitis, goose enteritis, goose infection. Myocarditis or goose abdominal hydronephritis. According to the age of infected goslings, the disease can be divided into three types: acute, subacute or chronic. Acute type can cause 100% death of goslings within 10 days of age. The gosling plague virus is mainly transmitted through the secretions, excretions, eggs, and water, feed, utensils, and venues of sick geese.
  • the spirit is stunned, out of the group, the nostrils flow out of serous nasal fluid, the goose frequently shakes the head, pulls grayish yellow or yellow-green dilute feces, nervous disorder, the mucosal necrosis of the posterior segment of the small intestine and the fibrinous exudate coagulate to form a suppository.
  • the shape is shaped like a sausage.
  • septicemia, high morbidity and mortality is extremely harmful to the production of goose industry.
  • the disease mainly occurs in goslings and young muscovy ducks of 1 to 3 weeks old, especially when it is about 1 week old. After 4 weeks of age, goose or muscovy ducks rarely show clinical symptoms.
  • the object of the present invention is to provide a muscovy duck parvovirus subunit vaccine, which provides the advantages of high efficiency, good safety and high protection rate, thereby compensating for the deficiencies of the prior art.
  • the Muscovy duck parvovirus subunit vaccine of the present invention comprises an antigen and a vaccine adjuvant, and the antigen used is Muscovy duck parvovirus VP protein, wherein the VP protein comprises:
  • amino acid sequence is the protein of SEQ ID NO: 1
  • the VP protein is inactivated by a formaldehyde solution
  • the content of the VP protein in the above vaccine is not less than 150 ⁇ g/ml.
  • the adjuvant used in the above inactivated vaccine is an aluminum hydroxide gel adjuvant.
  • the vaccine prepared by the invention can improve the antibody level of Muscovy duck after immunizing Muscovy duck, and ensure the maternal resistance of the progeny Body level, prevention of muscovy duck goose plague virus infection caused by muscovy duck goose plague virus; immunization 1 day old muscovy duck can effectively prevent the infection of young muscovy duck goose plague virus caused by muscovy duck goose plague virus.
  • Figure 1 Figure of the results of BlastP of the VP protein screened in the present invention.
  • the typical muscovy duck virus disease symptoms appeared in the young ducks in a duck farm.
  • the ducks that had developed the disease had previously been injected with the muscovy duck parvovirus vaccine. Therefore, it is suspected that the gene mutation occurred in the pathogenic bacteria.
  • the immune effect of the vaccine is not good.
  • the liver, spleen and pancreas of the dead duck were aseptically collected, and the suspension was homogenized with sterile physiological saline to prepare a suspension. The bacteria were removed by centrifugation and then inoculated into the 11-day-old Muscovy duck embryo through the allantoic cavity.
  • the purified virus liquid was purified and analyzed for virus characteristics, immunogenicity, specificity and purity. The results showed that the isolated strain reacted specifically with Muscovy parvovirus, without bacteria and mycoplasma. And foreign virus contamination.
  • the purified virus strain was named GPV201401.
  • the traits of the strains screened by the present invention were tested, and the results showed that the strain was a parvovirus.
  • the results of the challenge experiments indicated that the virus was selected to cause diarrhea and partial duck duck mucosa detachment to form embolism.
  • the vaccine prepared by the strain is immunized with 1 day old muscovy duck, and the antibody can be produced in 10 days, and the immunization period can be more than 6 months. After immunizing adult muscovy duck, it can protect the offspring of the muscovy duck within 6 months of immunization. Attacked by popular strains.
  • the structural protein gene VP of the GPV201401 strain was screened and identified.
  • the VP gene was 2199 bp in length and encoded 732 amino acids.
  • the VP gene sequence in GenBank was genetically evolved (Fig. 1) and nucleotide sequence. Based on the analysis, it was found that the VP gene homology of the VP gene of GPV201401 strain and the other 30 GPV strains was between 85.9% and 90.3%. The results showed that the VP protein of the virus was screened and the reported gosling virus was reported. There are amino acid differences in the VP gene.
  • the DNAStar software was used to design primers to amplify the entire sequence of the VP gene, wherein the 5' end of the forward primer was added with a BamHI restriction site; A NotI restriction site was added to the 3' end of the reverse primer.
  • PCR with GPV201401 virus as template Amplify and recover the gene of interest using a DNA purification and recovery kit.
  • the recombinant plasmid pET32a-VP was obtained by using pET32a(+) plasmid, and the recombinant plasmid was transferred into the competent E. coli host strain, and a single clone was picked. The positive clone was identified by enzyme digestion, shaken and sequenced.
  • Example 3 Expression, purification, renaturation and SDS-PAGE identification of recombinant fusion protein
  • a single cloned recombinant plasmid with the correct sequencing result was transformed into an expression-bearing Rosetta host strain to construct a recombinant genetic engineering strain.
  • Purification and renaturation were carried out according to the following steps: (1) The bacterial solution for inducing expression was taken out and packed into a 50 ml centrifuge tube, centrifuged at 4000 r/min for 10 min, and the supernatant was discarded; (2) 1 ml of TE1 (Tris-cl 10 mM/L) was used.
  • the protein solution in the semi-permeable membrane bag was boiled for 10 min by 1:1 plus 2 ⁇ loading Buffer, and the supernatant was centrifuged, and the supernatant was identified by 12% SDS-PAGE electrophoresis to obtain the target protein.
  • the purified VP protein solution is inactivated with formaldehyde, and then mixed with aluminum hydroxide gel adjuvant at a volume ratio of 1:3 to prepare a genetic engineering subunit vaccine, which is equivalent to containing each milliliter of aluminum hydroxide rubber seedling
  • the fusion protein was 200 ⁇ g/ml.
  • the challenge of the vaccine of the present invention against the GPV201401 strain showed that the vaccine prepared by the GPV201401 strain had the best immunological effect, which was presumably caused by the mutation of the gene of the GPV201401 strain.

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Abstract

提供了一种番鸭细小病毒亚单位疫苗，包括抗原和疫苗佐剂。其所用的抗原为番鸭细小病毒VP蛋白，其氨基酸序列为SEQ ID NO：1。

Description

一种番鸭细小病毒亚单位疫苗 技术领域
本发明属于禽类疫苗制备技术领域，具体涉及一种番鸭细小病毒亚单位灭活疫苗。
背景技术
细小病毒病是鹅和番鸭(又称瘤头鸭)的高度传染性疾病,它又称为德兹西氏病、鹅流感、鹅瘟或小鹅瘟、鹅肝炎、鹅肠炎、鹅传染l生心肌炎或鹅腹水性肝肾炎。根据受感染小鹅的龄期不同,这病可分为急性、亚急性或慢性三个型。急性型可引起10日龄以内的雏鹅100％死亡。小鹅瘟病毒主要通过病鹅的分泌物、排泄物、种蛋及病鹅饮过的水、饲料、用具、场地等传播。临床以精神委顿，离群独偶，鼻孔流出浆液性鼻液，患鹅频频摇头，拉灰黄色或黄绿色稀粪，神经紊乱，小肠中后段黏膜坏死脱落与纤维素性渗出物凝固形成栓子，形如腊肠状为特征。常呈败血经过，发病率和死亡率很高，对养鹅业生产危害极大。该病主要发生于1～3周龄的雏鹅和雏番鸭，尤其是1周龄左右的更易感，4周龄以上的鹅或番鸭感染后，很少表现临床症状。本病最早于20世纪60年代中后期出现于中国和许多欧洲国家，直到1978年才将该病称为鹅细小病毒感染，但是通过病毒中和试验、分子生物学研究证明，从鹅和番鸭分离到的细小病毒有明显的差异。如果对死亡的番鸭处理不当，往往造成病毒的蔓延，产生更大的危害。
发明内容
本发明的目的是提供一种番鸭细小病毒亚单位疫苗，所提供的疫苗具有高效、安全性好、保护率高的优点，从而弥补现有技术的不足。
本发明的一种番鸭细小病毒亚单位疫苗，包括有抗原和疫苗佐剂，其所用的抗原为番鸭细小病毒VP蛋白，其中VP蛋白包含有：
1)氨基酸序列为SEQ ID NO:1的蛋白，
2)在1)中限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有1)中蛋白活性，由1)衍生的蛋白。
编码上述VP蛋白的基因，其核苷酸序列为SEQ ID NO:2；
其中的VP蛋白经过甲醛溶液灭活；
上述的疫苗中VP蛋白的含量不低于150μg/ml。
上述灭活疫苗所使用的佐剂为氢氧化铝胶佐剂。
本发明制备的疫苗免疫番鸭后能提高番鸭的抗体水平，保证其子代母源抗 体水平，预防番鸭源小鹅瘟病毒引起的雏番鸭小鹅瘟病毒感染；免疫1日龄雏番鸭能有效预防番鸭源小鹅瘟病毒引起的雏番鸭小鹅瘟病毒感染。
附图说明
图1：本发明筛选的VP蛋白的BlastP的结果图。
具体实施方式
下面结合具体实施例对本发明进行详细的描述。本发明所应用的方法可以采用疫苗制备领域中常用的方法，而不仅限于本发明实施例的具体记载，本领域的普通技术人员可以其它常规方法来实现本发明。
实施例1、番鸭细小病毒VP蛋白的筛选
2014年某鸭场的雏番鸭群中出现了典型的番鸭细小病毒病症状，但发病的鸭群之前已经注射过番鸭细小病毒病疫苗，因此，怀疑是致病菌发生了基因突变，导致疫苗的免疫效果不佳。为了分离病源，无菌采集濒死鸭的肝脏、脾脏及胰腺，用无菌生理盐水匀浆制成悬液，离心取上清除菌后经尿囊腔分别接种11日龄番鸭胚，孵化168小时，收集24小时后死亡胚的尿囊液及胚体组织，经匀浆后，反复冻融后，取上清液冻存。收获的病毒液经纯化后进行了病毒含量、免疫原性、特异性及纯净性等方面的病毒特性的分析检测，结果表明分离的毒株与番鸭细小病毒发生特异性反应，无细菌、支原体及外源病毒污染。纯化后的病毒株命名为GPV201401。
对于本发明筛选的毒株的性状进行检测，结果表明，该株病毒属细小病毒，攻毒实验结果表明筛选的该病毒可引起雏番鸭发生以腹泻、部分鸭肠粘膜脱落形成栓塞为特征的番鸭细小病毒病。该毒株制备的疫苗免疫1日龄雏番鸭，10天就可产生抗体，免疫期可达6个月以上；免疫成年番鸭后，能保护免疫6个月内种番鸭所产子代免受流行毒株的攻击。
对筛选鉴定的GPV201401株的结构蛋白基因VP进行了测序,VP基因全长2199bp，编码732个氨基酸；将其与GenBank中收录的VP基因序列进行遗传进化树(图1)及核苷酸序列同源性分析，发现本发明获得GPV201401株VP基因与另外30个GPV毒株的VP基因同源性介于85.9％～90.3％之间；结果表明筛选病毒的VP蛋白与已报道的小鹅瘟病毒VP基因存在着氨基酸差异。
实施例2表达VP基因的重组质粒的构建
根据VP基因序列与表达性载体PET32a(+)的多克隆酶切位点，利用DNAStar软件设计引物来扩增VP基因的全序列，其中正向引物的5′端加入了BamHⅠ酶切位点；反向引物的3′端加入了NotⅠ酶切位点。以GPV201401病毒为模板进行PCR 扩增，并用DNA纯化回收试剂盒回收目的基因。利用pET32a(+)质粒获得pET32a-VP重组质粒，将该重组质粒转入表达性大肠杆菌宿主菌,挑取单个克隆，酶切法鉴定阳性克隆，摇菌，测序。
实施例3.重组融合蛋白的表达、纯化、复性及SDS-PAGE鉴定
将测序结果正确的单个克隆重组质粒转化表达性Rosetta宿主菌构建出重组基因工程菌。将该重组基因工程菌接种于含有氨苄青霉素(100mg/L)的LB细菌培养基,37℃振摇(200r/min)培养过夜，次日取出1ml培养物接种于120ml的LB培养基中，37℃振摇(200r/min)培养2.5h后，菌液摇至半透明半浑浊状态，吸光光度法测得OD600＝0.6～0.8,在诱导前先取10ml作为诱导前对照，并将振摇温度变为30℃加α-乳糖(终浓度为1.0mmol/L)诱导，分别诱导1h、2h、3h、4h、5h、6h收集10ml菌液，以诱导未转化pET32a(+)载体的菌液作为空白对照，以诱导转化pET32a(+)载体的菌液作为空载体对照。按照以下步骤进行纯化、复性：(1)将诱导表达的菌液取出分装至50ml离心管中，4000r/min离心10min，弃上清；(2)用1ml TE1(Tris-cl 10mM/L,EDTA二钠1mM/L,PH＝8.0)重悬沉淀并转移至1.5ml离心管中，按照以下条件：超声30s、间隔30s、工作20次、480V电压下超声波裂解破碎工程菌，7000r/min 4℃离心20min，弃上清；(3)将沉淀中加入1ml TE2(Triton X-100/TE1＝1/100配置)震荡混匀，7000r/min 4℃离心20min，弃上清；(4)在沉淀中加入1ml TE3(用TE1配置2M/L尿素)，7000r/min4℃离心20min，弃上清；(5)将沉淀收集加入150ml变性液(8M/L尿素、10mM/L Tris-cl、10mM/L DTT)搅拌溶解，8000r/min 4℃离心30min，取上清；(6)将上清液加入半透膜袋中，4℃透析复性48h。取半透膜袋中的蛋白液按1:1加2×上样Buffer煮沸10min，离心取上清，上清用12％SDS-PAGE电泳鉴定分析获得目的蛋白。
将纯化好的VP蛋白溶液用甲醛灭活后，与氢氧化铝胶佐剂按体积比＝1：3进行配苗，制备成基因工程亚单位疫苗，相当于每毫升氢氧化铝胶苗中含有融合蛋白200μg/ml。
实施例4、疫苗效力检验
1)取1日龄雏番鸭20只，随机分成两组，每组10只，其中一组每只颈部皮下注射疫苗，0.2ml/只，另一组10只同日龄雏番鸭不免疫作对照，隔离饲养。于免疫后15日免疫组和对照组同时攻击雏鸭源小鹅瘟病毒GPV201401株，每只颈部皮下注射疫苗，0.2ml/只(106.50ELD50/0.2ml)，连续观察15日，结果免疫组鸭9只保护，对照组鸭10只全部发病。(见表1)。
表1：疫苗效力检验结果
Figure PCTCN2016107376-appb-000001
2)子代母源抗体与攻毒保护的关系90日龄种番鸭20只，每只颈部皮下注射疫苗，2.0ml/只，另取10只同日龄种番鸭不免疫作对照。于免疫后15日相同途径相同剂量再免疫一次，收集开产后4月时所产种蛋孵化出雏，于雏番鸭7日龄时，免疫组子代取10只，对照组子代取10只，攻毒GPV201401株，每只肌肉注射0.2ml。免疫组子代保护9只，对照组全部发病。
申请人将番鸭的数目扩大，再次进行免疫；结果表明本发明制备的疫苗对番鸭细小病毒具有很好的免疫效果，数据具有统计学的意义。
3)对地方流行毒株的攻毒保护(毒株的选育)90日龄种番鸭20只，每只颈部皮下注射疫苗，2.0ml/只，另取10只同日龄种番鸭不免疫作对照。于免疫后15日相同途径相同剂量再免疫一次，收集开产1个月内所产种蛋孵化出雏，于雏番鸭7日龄时，免疫组子代取10只，对照组子代取10只，攻毒6株各地方分离株，每只肌肉注射0.2ml。结果表明，用GPV201401株制备的番鸭小鹅瘟灭活疫苗相比较其它毒株制备的灭活疫苗，能抵御各地方分离毒的攻击。
此外，本发明的疫苗对于GPV201401株的攻毒实验表明，GPV201401株制备的疫苗的免疫效果最好，推测是由于GPV201401株基因发生变异导致的。
上述结果表明，本发明制备的疫苗能有效的预防目前各地流行的番鸭小鹅瘟的感染，具有很好的应用前景。

Claims (5)

  1. 一种番鸭细小病毒亚单位疫苗，其特征在于，所述的疫苗包含有抗原和疫苗佐剂，所用的抗原为番鸭细小病毒VP蛋白，其中VP蛋白包含有：
    1)氨基酸序列为SEQ ID NO:1的蛋白，
    2)在1)中限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有1)中蛋白活性，由1)衍生的蛋白。
  2. 如权利要求1所述的番鸭细小病毒亚单位疫苗，其特征在于，所述的VP蛋白的编码基因，其核苷酸序列为SEQ ID NO:2。
  3. 如权利要求1所述的番鸭细小病毒亚单位疫苗，其特征在于，所述的VP蛋白经过甲醛溶液灭活。
  4. 如权利要求1所述的番鸭细小病毒亚单位疫苗，其特征在于，所述的VP蛋白的含量不低于150μg/ml。
  5. 如权利要求1所述的番鸭细小病毒亚单位疫苗，其特征在于，所述的疫苗佐剂为氢氧化铝胶佐剂。
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