In-vitro expression method of pig Six1 protein and preparation method of polyclonal antibodies of pig Six1 protein

An in vitro expression and protein expression technology, applied in the field of genetic engineering, can solve the problems of good specificity and high titer, and achieve the effect of good specificity and high potency

Inactive Publication Date: 2015-01-07
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Sixl is an important regulator of muscle fiber type and a potential factor affecting pork quality. At present, there are many Sixl antibodies on the market for humans, mice and rats. These antibodies have certain cross-reactions with pigs, cattle, sheep, rabbits, monkeys, etc. , but there is no antibody with high titer and good specificity against porcine Sixl

Method used

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  • In-vitro expression method of pig Six1 protein and preparation method of polyclonal antibodies of pig Six1 protein
  • In-vitro expression method of pig Six1 protein and preparation method of polyclonal antibodies of pig Six1 protein
  • In-vitro expression method of pig Six1 protein and preparation method of polyclonal antibodies of pig Six1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction and Identification of Escherichia coli Expression Vector pET30a(+)-pSix1

[0036] Take the longissimus dorsi muscle of normal pigs, extract the total RNA of longissimus dorsi with the TRIzol kit, reverse transcribe with the reverse transcription kit to obtain the total cDNA, and use this as a template to carry out PCR amplification, PCR reaction system (total reaction system 50μl) for: ddH 2 O20 μl, 1 μl of upstream and downstream primers (10 μm), 3 μl of cDNA, 25 μl of 2×Taq PCR master mix. The PCR reaction conditions were: pre-denaturation at 95°C for 3 minutes, followed by 30 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, for a total of 35 cycles, and finally extension at 72°C for 10 min. PCR products were identified by 1% agarose gel electrophoresis. Depend on figure 1It can be seen that there is a clear bright band between 750 and 1000 bp, which is consistent with the expected size.

[0037] The PCR products were recovered u...

Embodiment 2

[0042] Example 2: Expression of pig Six1 in Escherichia coli

[0043] 1. Obtain the genetically engineered bacteria expressing pig Six1: inoculate the successfully sequenced bacteria into 4 ml of LB liquid medium containing kanamycin (50 μg / ml), shake and culture overnight at 37° C. and 250 rpm, and follow the E.Z.N.A plasmid of OMEGA Company Extract the pET30a(+)-pSix1 recombinant expression vector with the mini-extraction kit and transform it into Escherichia coli strain BL21(DE3) competent cells. The recombinants were cultivated and screened, and the obtained recombinants were genetically engineered bacteria expressing pig Six1. Randomly select a single colony for streak culture. After 12 hours, take a small amount of the streak culture bacteria that grew out and inoculate them in 4ml LB liquid medium (containing 50μg / ml kanamycin), shake culture at 37°C and 250rpm overnight, and press 850μl Add 150 μl sterilized glycerin to the bacterial solution, and mix well to obtain g...

Embodiment 3

[0051] Example 3: Detection, separation, purification and identification of the expression form of the recombinant porcine Six1 protein

[0052] 1. Detection of expression form of recombinant porcine Six1 protein

[0053] Inoculate the activated engineered bacteria into 50ml LB liquid medium (containing 50μg / ml kanamycin), culture at 37°C, shake at 250rpm until OD 600 Add IPTG when it is 0.4 to 0.6, according to the results of the exploration of the induction conditions (such as figure 2 , image 3 ), so that the final concentration was 2.0mmol / L, centrifuged after 2 hours of induction at 30°C to collect the precipitate, resuspended the bacteria with 6mL0.01mol / LPBS (pH7.4) and then ultrasonically disrupted: the power was 260W, ultrasonication for 2 seconds, Interval 5 seconds, act for 30 minutes until the solution is clear, centrifuge at 1200rpm for 30 minutes, save the supernatant and precipitate (inclusion body) respectively, and perform SDS-PAGE electrophoresis, stain w...

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Abstract

The invention discloses an in-vitro expression method of pig Six1 protein and a preparation method of polyclonal antibodies of the pig Six1 protein. The preparation method comprises the following steps: cloning a pig Six1 gene through primer design; constructing an escherichia coli recombinant expression vector pET-30a(+)-pSix1; transferring the constructed recombinant expression vector pET-30a(+)-pSix1 into the escherichia coli BL21(DE3) to construct and express a recombinant strain of the pig Sixl protein; optimizing the induction expression conditions of the recombinant strain and purifying the recombinant pig Six1 protein; and preparing the pig Six1 polyclonal antibodies according to a conventional polyclonal antibody preparation method with the purified recombinant pig Six1 protein as the antigen. According to the method disclosed by the invention, the in vitro expression of the pig Six1 protein is achieved and the pig Sixl polyclonal antibodies with high tier and good specificity are prepared, and therefore the requirements of relevant experiments are met.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an in vitro expression of pig Six1 protein and a preparation method of polyclonal antibody thereof. Background technique [0002] In recent years, the pursuit of rapid growth rate and lean meat percentage in the pig breeding process has led to a decline in pork quality, which does not meet the growing meat quality requirements of consumers. The type of muscle fiber is closely related to the quality of pork, and the slow oxidation type muscle fiber contributes to the tenderness and juiciness of pork. Therefore, increasing the proportion of slow oxidative muscle fibers in muscle can effectively improve pork quality. [0003] Sixl is an important member of the Six transcription factor family, which plays an important role in early myogenic cell determination and migration, myofiber formation and myocyte proliferation in skeletal muscle. It can act as a transcriptional ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/47C07K16/18
Inventor 黄志清陈小玲徐孟陈代文余冰
Owner SICHUAN AGRI UNIV
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