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

A technology of Peste des petits ruminants and reaction, applied in the field of molecular biology, can solve problems such as difficult synthesis, time-consuming and labor-intensive, complex secondary structure, etc.

Active Publication Date: 2012-12-19
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

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The transformation of embodiment 1 PPRVN protein gene:

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

[0038] ATGGCTACTTTATTAAAATCTTTAGCTTTATTTAAACGTAATAAAGATAAAGCTCCTACTGCTTCTGGTTCTGGTGGTGCTATTCGTGGTATTAAAAATGTTATTATTGTTCCTATTCCTGGTGATTCTTCTATTACTACTCGTTCTCGTTTA...

Embodiment 2

[0039] Example 2 Synthesis of N gene by PCR

[0040] Materials: 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 company

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

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

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

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

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

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

[0051] 1. Primer Design

[0052] Primers were designed using the mo...

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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 petits ruminants virus (PPRV). Background technique [0002] Peste des petits ruminants (PPR) caused by Peste des petits ruminants virus (PPRV) is a severe contact with rinderpest-like symptoms that mainly occurs in goats, sheep and some wild small ruminants Class A infectious disease. Since the disease was first recorded in Ivory Coast in 1940, it has occurred in some countries in Africa and the Middle East. [0003] PPRV belongs to Paramyxoviridae and Morbillivirus. Other members of the same genus are rinderpest virus (RPV), canine distemper virus (CDV), seal distemper virus (PDV), dolphin distemper virus (DDV), bovine measles virus (MV2K1) and human measles virus (MV). PPRV is currently divided into 4 groups, of which 3 groups originated from Africa and one group originated from Asia. At present, only one serotype of...

Claims

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Application Information

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Patent Type & Authority Patents(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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