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Suspension PK15 cell line for expressing pig CD163 and CD169 molecules

A technology of CD169 and cell lines, applied in the field of transgenic technology, can solve the problems of low titer, unsuitable for subculture PRRSV, low sensitivity of PRRSV, etc., and achieve the effect of reducing production costs

Pending Publication Date: 2020-07-28
成都史纪生物制药有限公司
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AI Technical Summary

Problems solved by technology

Porcine alveolar macrophages (PAM) are the main target cells of PRRSV, but PAM cells are not suitable for subculture of PRRSV. The viruses used in PRRSV vaccines are all from African green monkey kidney cells Marc145 cultured, but the PRRSV virus effect of Marc145 cells cultured The price is low, and it can only be cultured on the wall or microcarrier suspension, which has high requirements for PRRSV scale-up production and cost control
[0004] PK15 cells, a kind of pig kidney cells, can be applied to the culture of some porcine viruses (such as porcine circovirus type 2), but they have low sensitivity to PRRSV and are also not suitable for PRRSV scale-up culture

Method used

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  • Suspension PK15 cell line for expressing pig CD163 and CD169 molecules
  • Suspension PK15 cell line for expressing pig CD163 and CD169 molecules
  • Suspension PK15 cell line for expressing pig CD163 and CD169 molecules

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Embodiment 1

[0033] Example 1 Construction and Identification of the Suspension PK15 Cell Line Expressing Porcine CD163 and CD169 Molecules of the Present Invention

[0034] 1. Obtain CD163 and CD169 genes

[0035] 1. Look up the mRNA sequences of CD163 (serial number: EU016226.1) and CD169 (serial number: AF509585.1) genes through the NCBI website. The primer sequences CD163F (SEQ ID NO.1) and CD163R (SEQ ID NO.2) were designed for amplifying the CD163 sequence; the primer sequences CD169F (SEQ ID NO.3) and CD169R (SEQ ID NO.4).

[0036] 2. Take 2 mL of cultured porcine primary alveolar macrophages, use Tiangen Biochemical Technology (Beijing) Co., Ltd. Animal Tissue Total RNA Extraction Kit (DP431) to extract total RNA, and then use RT-PCR kit (Tiangen Biochemical : KR118) was reverse transcribed into cDNA.

[0037] 3. The obtained cDNA was used as a template, and the CD163 gene was amplified by PCR with primers CD163F (SEQ ID NO.1) and CD163R (SEQ ID NO.2), CD169F (SEQ ID NO.3) and CD...

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Abstract

The invention discloses a suspension PK15 cell line for expressing pig CD163 and CD169 molecules and a preparation method thereof, and belongs to the technical field of transgenosis. The method comprises the following steps: 1) constructing recombinant plasmids; 2) transfecting PK15 cells by using lipidosome; 3) screening puromycin; and 4) performing suspension culture and domestication by using aserum-free culture medium. The cell line prepared by the method is very sensitive to porcine reproductive and respiratory syndrome viruses, can be used for amplifying and reproducing a large number of porcine reproductive and respiratory syndrome viruses, is low in cost, and has a good application prospect in vaccine production practice.

Description

technical field [0001] The invention belongs to the field of transgenic technology. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is an epidemic disease of porcine caused by porcine reproductive and respiratory syndrome virus (PRRSV), and its main symptoms are reproductive impairment and lung inflammation in pregnant sows. The disease was introduced into China in 1996 and broke out and became popular all over the country. Due to the rapid genetic variation and recombination of PRRSV, the disease has now become a major disease worldwide, causing very large economic losses to pig farming. [0003] In classification, PRRSV belongs to the genus Arterivirus of the family Arteriviridae, which is a positive-strand RNA virus with an envelope. PRRSV particles are spherical, with a diameter of about 50-65nm, and the nucleocapsid is icosahedral symmetry, with a diameter of about 30-35nm. PRRSV has a very strict cell tropism and mainly infects monoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/12C12N7/00
CPCC12N5/0686C07K14/70596C12N15/85C12N7/00C12N2510/02C12N2800/107C12N2770/10051
Inventor 方华明张冬冬欧仁芳刘小方潘倩
Owner 成都史纪生物制药有限公司
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