Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof

A PPR reaction technology, applied in the field of molecular biology, can solve problems such as difficult synthesis, complex secondary structure, time-consuming and labor-intensive problems

Active Publication Date: 2011-06-29
杨凌金海生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Then further connect and synthesize longer fragments, which is time-consuming and laborious, and fragments with complex secondary structures and high G\C content are not easy to synthesize

Method used

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  • Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof
  • Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof
  • Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Modification of PPRVN protein gene:

[0037] The amino acid synonymous modification was carried out according to the gene sequence of the tibet strain of the NCBI Peste des ruminants virus (Genbank accession number EU360596). The principle of the transformation is to make it suitable for expression in the prokaryotic system, using high-frequency ACC, CTG, CGT, ATC instead of ACA, CTA, AGA, ATA. The Arg codons AGA, AGG, CGG, CGA, Ile codon AUA, Leu codon CUA, Gly codon GGA and Pro codon CCC should be replaced as synonymously as possible. At the same time, considering that the endonuclease site that conflicts with the restriction endonuclease on the multiple cloning site of the expression vector cannot be introduced into the gene, the modified N protein nucleic acid sequence is as SEQ ID NO: 41:

[0038] ATGGCTACTTTATTAAAATCTTTAGCTTTATTTAAACGTAATAAAGATAAAGCTCCTACTGCTTCTGGTTCTGGTGGTGCTATTCGTGGTATTAAAAATG TTATTATTGTTCCTATTCCTGGTGATTCTTCTATTACTACTCGTTCTCGTTTATTAGATCGTT...

Embodiment 2

[0039] Example 2 Synthesis of N gene by PCR

[0040] Material: The N protein nucleic acid sequence comes from the modified gene sequence.

[0041] The primer sequence SEQ ID NO: 1-40 was synthesized by Shanghai Jierui Biological Co., Ltd.

[0042] Kit: PrimeSTAR HS DNA Polymerase Kit (Takara, catalog number DR010A)

[0043] pM D19-T simple vector (Takara, catalog number D104A)

[0044] PCR instrument (S1000 thermal cycler) was purchased from Bio Rad

[0045] Agarose was purchased from GENETECH (SHANGHAI) Co., Ltd. (batch 111760)

[0046] pET-32a is kept by our laboratory

[0047] Bl21(DE3)Plyss (preserved in this laboratory)

[0048] N protein monoclonal antibody is kept by our laboratory

[0049] The secondary antibody is HRP goat anti-mouse IgG antibody (LP1002a) purchased from Abgent Primary Antibody

[0050] Pre-stained protein Marker (SM0671) was purchased from Fermentas

[0051] 1. Primer design

[0052] Primers were designed using the modified gene sequence as a design template. A total...

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Abstract

The invention provides a modified coded nucleic acid of peste des petits ruminants virus N protein which has a sequence of SEQ ID NO:41, and also provides its PCR amplification method and a kit used for the method.

Description

Technical field [0001] The invention relates to the field of molecular biology, in particular to the modification and synthesis of the N gene of Peste des petitsruminants virus (PPRV). Background technique [0002] Peste des petits ruminants (PPR) caused by Peste des petits ruminants virus (PPRV) is a kind of severe contact with rinderpest symptoms that mainly occurs in goats, sheep and some wild small ruminants. Class A infectious disease. Since the disease was first described in Ivory Coast in 1940, it has occurred in some countries in Africa and the Middle East. [0003] PPRV belongs to the Paramyxoviridae family and the measles virus genus. Other members of the same genus include rinderpest virus (RPV), canine distemper virus (CDV), seal plague virus (PDV), dolphin plague virus (DDV), bovine measles virus (MV2K1) and human measles virus (MV). PPRV is currently divided into 4 groups, of which 3 groups originate from Africa and one group originates from Asia. Currently, only 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N15/10C12N15/63C12N1/21
Inventor 张国瑞李震雷志海董世娟朱玉敏于瑞嵩沈世缘王茜张西营叶小飞
Owner 杨凌金海生物技术有限公司
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