Preparation method and use of duck-derived coronavirus attenuated strain DCV35

A technology for coronaviruses and attenuated strains, applied in antiviral agents, medical preparations containing active ingredients, inactivation/attenuation, etc., can solve the problems that IBVZZ2004 cannot produce effective protection and low affinity

Inactive Publication Date: 2015-07-08
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The IBVZZ2004 strain has a very low affinity with the domestic popular IBV, and also has a low affinity with the main domestic vaccine strains, such as the homology of M41 strain, H120, Beaudette, and IBV4/91 is 85.2%~87.9%
The isolation and identification methods, pathogenic

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Induction of Attenuated Strain by Low Temperature Culture

[0014] ZZV6 seed poisoning (EID 50 for 10 -4.88 / 0.2mL) first passed through a 56°C water bath for 20min, filtered with a 0.22um bacterial filter, and inoculated with 9-day-old SPF chicken embryos, 5 eggs per generation, 0.2mL per embryo, and incubated at 31-32°C. Daily observation, chicken embryos that died within 24 hours were discarded, and the dead chicken embryos were temporarily placed in a refrigerator at 4°C, and the virus was aseptically collected for 72 hours. At the same time, the pathological changes of the chicken embryos were observed, and the allantoic fluid of typical diseased chicken embryos was selected for passage , each generation was treated and cultured in the same way, continuously passed to the 25th generation, cultured at 37°C from the 26th generation, and then continuously passed to the 35th generation to obtain DCV35.

Embodiment 2

[0015] Example 2 Determination of the sequence of the S1 gene of the passaged virus

[0016] The 7th, 15th, and 20th to 35th passage viruses were selected during the continuous passage, and the S1 gene was cloned by RT-PCR to determine its nucleic acid sequence. S1 gene primers: upstream (P1): 5'-AGTTATTGGTTAGAGATGTTGGGGA-3', downstream (P2): 5'-C GTA TGG ACA GCT TGT GAC ATT TTC-3'.

[0017] Using the DNASTAR analysis software, the base sequence and deduced amino acid sequence of the sequenced 7th, 15th, 20th, 22-35th generation virus and the parental virus's S1 gene and the difference in the amino acid sequence are shown in Table 1. The results of the comparison and analysis of the bases and amino acids of each generation See Appendix A and Appendix B. The results showed that 12 bases of the S1 gene mutated in the 22nd generation, and this variation remained stable in the 35th generation. The 12 mutation sites of the S1 gene are: 188ntT→C (Val→Ala), 191ntA→G(Asn→Ser), 228nt...

Embodiment 3

[0019] Example 3 The pathogenicity test of DCV35 to SPF chicks

[0020] (1) DCV35 EID 50 Take DCV35 1mL and filter it with a 0.22um sterile filter, then make a 10-fold incremental dilution with normal saline, inoculate five 9-day-old SPF chicken embryos at each dilution, 0.2mL / each embryo, culture at 37°C, and observe every day The number of dead embryos was recorded, and the chicken embryos that died within 24 hours were discarded, and the remaining live embryos were dissected after incubation until 19 days old, and the dead and dwarf embryos were used as the criteria for judging infection. Nine-day-old SPF chicken embryos were inoculated with the same dose of normal saline as a control. Calculate EID by Reed-Muench method 50 .

[0021] The results showed that: with the increase of the number of passages of the virus, the adaptability of the virus to SPF chicken embryos became stronger and stronger, and EID 50 It also increased accordingly, and the EID of DCV35 was calcul...

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Abstract

The invention relates to a preparation method and use of a duck-derived coronavirus attenuated strain DCV35. The preparation method is characterized in that a duck-derived coronavirus virulent strain IBV ZZ2004 (with microbial preservation number of CGMCC NO.3842) separated by the inventor is subjected to passage attenuation, the S1 gene sequence of the screened attenuated strain DCV35 has obvious characteristics, and the variant sequence has stable heredity in 22th-35th generations. In SPF chicken infection adopting an artificial infection test and a horizontal transmission test, the screened attenuated strain DCV35 does not produce virulence enhancement. An experiment proves that after inoculation of a chicken with the attenuated strain, the attenuated strain can effectively activate a chicken immunization system, has good effects of preventing chicken infectious bronchitis and can be used as a vaccine for preventing chicken infectious bronchitis.

Description

technical field [0001] The invention relates to the preparation and application of a duck-derived coronavirus (Duck Coronavirus, DCV) attenuated strain DCV35, belonging to the field of biotechnology. Background technique [0002] Chicken infectious bronchitis virus (Infectious bronchitis virus, IBV) seriously endangers the breeding industry in our country, and the economic loss caused by chicken infectious bronchitis is very serious every year. The main method of preventing this disease is attenuated vaccine, which is the most widely used The most attenuated vaccines are H120 and H52. However, due to the large number of IBV serotypes and the easy mutation of the IBV genome, new IB mutant strains continue to emerge, making the existing vaccines unable to produce effective protection against the newly emerging mutant epidemic strains, making the prevention and treatment of IBV difficult. [0003] IBV ZZ2004 is a virus isolated from muscovy ducks that cause immunosuppression a...

Claims

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Application Information

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IPC IPC(8): C12N7/08A61K39/215A61P31/14C12R1/93
Inventor 刘兴友陈明艳姚四新欧长波刘明成
Owner HENAN INST OF SCI & TECH
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