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Method for preparing swine novel gene BCL-GL polyclonal antibody

A polyclonal antibody, gene technology, applied in the field of bioengineering

Inactive Publication Date: 2011-02-02
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether similar genes are contained in pigs has not yet been reported.

Method used

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  • Method for preparing swine novel gene BCL-GL polyclonal antibody
  • Method for preparing swine novel gene BCL-GL polyclonal antibody
  • Method for preparing swine novel gene BCL-GL polyclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0024] porcine BCL-G L Gene cloning:

[0025] human BCL-G L The gene cDNA sequence (accession number NM_138722) is the seed sequence, and the pig ESTs database is BLAST aligned and spliced ​​in GenBank to obtain the pig BCL-G L cDNA sequence of the gene. Using this sequence as a reference, the upstream cloning primer (SEQ ID NO: 1) P1: 5'-TCCTTACTGCCACCTGAC-3', and the downstream cloning primer (SEQ ID NO: 2) P2: 5'-ATGCTGCCACATCCTATG-3' were designed.

[0026] Spleen tissue of 1-day-old Yorkshire pigs was collected, total RNA was extracted by TRIzol method, and total cDNA was obtained by reverse transcription with a reverse transcription kit. Using this as a template, PCR was performed, and porcine β-actin was used as an internal reference for PCR. The 25 μL reaction system includes: Premix Taq enzyme 12.5 μL, cDNA 1.25 μL, primer P1 0.5 μL, primer P2 0.5 μL, ddH 2 O 10.25 μL. The PCR parameters were 95°C for 5min, then 94°C for 1min, 53°C for 1min, 72°C for 1.5min, a t...

Embodiment 2

[0028] Prokaryotic expression vector pET32a-BCL-G L Construction: based on pMD19-BCL-G L Based on vector sequencing results, design primers for prokaryotic expression. Upstream primer (SEQ ID NO: 3) P3: 5'-AAGGATCCATGTGCACCACCAGC-3', downstream primer (SEQ ID NO: 4) P4: 5'-CCAAGCTTCAGTCTACTTCTTCATGGG-3'. Using the total cDNA as a template, PCR was performed with upstream and downstream prokaryotic expression primers. The 25 μL reaction system included: Premix Taq 12.5 μL, template 1.25 μL, primer P30.5 μL, primer P40.5 μL, ddH 2 O 10.25 μL. The PCR parameters were 95°C for 5min, then 94°C for 1min, 60°C for 1min, 72°C for 1.5min, a total of 30 cycles, and then 72°C for 10min. Amplify the target sequence. The target sequence and pET32-a vector were digested with BamH I and Hind III for 3 hours. Perform electrophoretic detection and gel recovery. The recovered product was ligated overnight at 16°C with T4 DNA ligase. The ligation product was transformed into DH5α competen...

Embodiment 3

[0030] Eukaryotic expression vector pEGFP-BCL-G L Construction of the eukaryotic expression vector: the construction method of the eukaryotic expression vector is basically the same as in Example 2, the upstream primer (SEQ ID NO: 5) P5: 5'-CAAAGCTTATATGTGCACCACCAGCACC-3', the downstream primer (SEQ ID NO: 6) P6: 5' - CCGGATCCTCAGTCTACTTCTTCATGGG-3'; the expression vector used was pEGFP-C1. The recombinant plasmid was named pEGFP-BCL-G L .

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Abstract

The invention discloses a method for preparing a swine novel gene BCL-GL polyclonal antibody. The method comprises the following steps of: A1, cloning a swine BCL-GL gene; A2, constructing a prokaryotic expression vector pET32-a-BCL-GL; A3, constructing a eukaryotic expression vector pEGFP-BCL-GL; A4, expressing a pronucleus of a BCL-GL protein; and A5, purifying the BCL-GL protein and preparing the BCL-GL polyclonal antibody against a Guinea pig.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a new pig gene BCL-G L Preparation method of polyclonal antibody. Background technique [0002] Apoptosis is also called "programmed cell death" or "cell suicide". In recent years, a lot of research work has revealed that apoptosis is as important as cell proliferation and differentiation. For the immune system, apoptosis is not only an essential link in the development of the immune system, but also a way for the immune system to perform its functions. More and more research data show that cell apoptosis is closely related to tumorigenesis and the removal of virus-infected cells, so the research on cell apoptosis is also of great significance to the treatment of tumors, viral diseases and other diseases. The occurrence and regulation of apoptosis involves multiple gene families, among which the BCL2 gene family is a gene family closely related to apoptosis, and members ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/12C07K16/06C07K14/47C07K16/18
Inventor 蒋朋飞张德礼
Owner NORTHWEST A & F UNIV
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