Duck-origin coronavirus N protein gene, clone method and application thereof
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A technology of coronavirus, cloning method, applied in the field of bioengineering
Inactive Publication Date: 2011-01-19
HENAN INST OF SCI & TECH
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The nucleoprotein is highly conserved within the group, but recent studies have shown that there are variations in the N gene among different strains, and these differences exist in putative epitopes and functionally related regions, suggesting that the N genes of different 1BV strains vary Changes in certain biological properties that may affect the virus
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Embodiment 1
[0023] The cloning method of the N gene of a kind of duck origin coronavirus ZZ2004 of embodiment 1 comprises the steps:
[0024] (1) PCR primer design Design a pair of specific primers, the sequence is as follows:
[0025] Upstream primer 5'-AGTCAGTAGACAGCGGAGAG-3';
[0026] Downstream primer 5'-TTCGTTTCCAGGCTACTAAG-3'.
[0027] (2) The enrichment method of ZZ2004 strain
[0028] Dilute the allantoic fluid infected by the ZZ2004 strain 1:10 with sterilized PBS, inoculate 0.2ml into the chorioallantoic cavity of 9-day-old SPF chicken embryos, incubate at 37°C for 36-48h, place at 4°C for 12h, and harvest the chicken Embryo allantoic fluid.
[0029] Take 50ml of SPF chick embryo allantoic fluid infected by ZZ2004 strain into three sterilized centrifuge tubes, centrifuge at 5000rpm for 20min; discard the precipitate respectively, take the supernatant in another sterilized centrifuge tube, centrifuge at 12000rpm for 20min; discard the precipitate separately , take the superna...
Embodiment 2
[0073] Example 2ZZ2004 strain N gene deduced amino acid sequence homology alignment, see figure 1 .
[0074] Using DNAstar software, the deduced amino acid sequence of the N gene of the ZZ2004 strain was compared with seven reference strains registered in GenBank. Compared with the classic strain Beaudette strain, the variation regions of the N protein of the strain are mainly concentrated in the regions of 5-8aa, 184-202aa, 220-247aa, 322-336aa, and some scattered point mutations in other regions; the homology rate is the highest Compared with the SAIBK strain, the difference is relatively small, only 6 amino acid changes, namely, 7 A → S, 64 T → N, 123 V → T, 223 I → M, 351 T → N , 357 bits R→Q.
[0075] genetic tree (see figure 2 ) analysis results showed that the isolate ZZ2004N protein and the domestic isolate SAIBK strain belonged to the same branch, and the genetic relationship with the SAIBK strain was the closest, and the genetic relationship with the standard str...
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Abstract
The invention relates to a N protein gene of duck-origin coronavirus ZZ2004, and a clone method and application thereof. The N protein gene sequence has the overall length of 1230 bp, and codes N proteins of 409 amino acid residues. The virus sequence with higher homology is found through random PCR, a pair of specific primers is designed by using the sequence as the template and the corresponding fragments are amplified, and then the fragments are respectively connected with a pMD18-T carrier to clone the N protein gene of the virus. Due to that the N gene is an important immune gene and has important effect in the aspects of cellular immunity, humoral immunity and local immunity. The research of the N gene of the invention favors the discovery of the IBV heteromorphosis regularity, the distinguishing of the different serotypes and the further development of novel genetic engineering vaccine, and plays important role in the effective prevention of the IBV.
Description
technical field [0001] The invention relates to the field of bioengineering, in particular to an N protein gene of a duck-derived coronavirus ZZ2004 and a cloning method and application thereof. Background technique [0002] In 1937, coronaviruses (Coronaviruses) were first isolated from chickens. In 1965, the first human coronavirus was isolated. It is named "coronavirus" because it can be observed under an electron microscope that there are obvious rod-shaped particle protrusions on its outer membrane, making it look like the crown of a medieval European emperor. According to the serological characteristics of the virus and the differences in nucleotide sequences, the Coronaviridae family is currently divided into two genera, coronavirus and torovirus. The representative strain of Coronaviridae is Avian infectious bronchitis virus (IBV). The virus is an enveloped, moderately pleomorphic spherical or oval virion with rod-shaped protrusions on the surface. [0003] Avian...
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