Expression purification method for HBcAg

An expression, purification and inducible expression technology, applied in the field of HBcAg expression and purification, can solve the problems of low expression amount and complicated purification method, and achieve the effect of simple steps, good antigenicity, and satisfying detection requirements.

Inactive Publication Date: 2018-10-16
GETEIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of eukaryotic expression system to express HBcAg is seldom, and there is a problem of low expression level
In addition, regarding the purificatio...

Method used

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  • Expression purification method for HBcAg
  • Expression purification method for HBcAg
  • Expression purification method for HBcAg

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Construction of HBc yeast multi-copy vector

[0061] The full-length HBc gene is obtained by codon optimization according to the C-terminal arginine-rich region of the gene C-type sequence published by NCBI. The sequence is shown in SEQ ID NO: 1. The whole gene fragment is chemically synthesized and then amplified by PCR. Gene.

[0062] The primers used in PCR amplification are:

[0063] N-terminal primer:

[0064] CCAATgaattcGCCACCATGGACATTGACCCGTATAAAG;

[0065] C-terminal primer:

[0066] CAATgaattcTTAACACTGGCTTTCGCGAGAC.

[0067] The HBcAg gene of the present invention is the domestic popular strain sequence (KR013930.1) found on NCBI, GCCACC is added to the N-terminus to facilitate the correct initiation of translation of the target gene, and codon optimization is performed at the C-terminus to facilitate protein expression.

[0068] After PCR amplification, the target gene was recovered by gel, EcoR I single-digested, and then the digested fragment was recove...

Embodiment 2

[0073] Transformation and Screening of Multi-copy Recombinants of HBc Yeast

[0074] Extraction and linearization of multi-copy recombinant plasmids: Take 20 μg of the correctly identified four-copy plasmid and digest it with Sal I. The digested product is recovered through a nucleic acid column, and precipitated with 1 / 10 volume of 3M NaAc and 2 times the volume of absolute ethanol. Wash with 70% ethanol and redissolve in 10 μl sterile water.

[0075] Yeast Competent Preparation: GS115 strain YPD plate was streaked, and when the single clone grew out, the single clone was picked in 50mLYPD medium, cultivated overnight, and the OD 600 When it reaches 1.2-1.6, centrifuge at 1500g 4°C for 5min, wash twice with pre-cooled sterile water, wash once with pre-cooled 1M sorbitol, resuspend in 1mL pre-cooled 1M sorbitol, take 100μl competent cells and add Transfer to the above-mentioned linearized plasmid, transfer to the electroporation cup, ice-bath for 5 minutes, transform by elect...

Embodiment 3

[0077] Induced expression of HBc yeast

[0078] For the correctly identified transformants, take part of the strains and inoculate them in BMGY medium at 30°C for 16-18 hours, then replace them with BMMY medium to make the OD 600 =0.8-1, supplemented with 0.5% methanol every 12 hours, and continuously induced for 4 days. After the expression is finished, take 20 μL of the bacterial liquid for SDS-PAGE identification, and the expression identification results are shown in figure 2 . Compared with the control, the multi-copy HBc recombinant yeast had obvious expression band at 22kD.

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Abstract

The invention relates to the field of genetic engineering and in particular relates to an expression purification method for hepatitis B core antigen (HBcAg). The expression purification method comprises the following steps: carrying out linearization on a yeast expression vector with a nucleotide sequence for expressing HBcAg, and transinfecting competence yeast so as to obtain a transformant; carrying out amplified culture and induced expression on the transformant, crushing a cell, centrifuging, and collecting supernate; carrying out fractional precipitation and anion exchange chromatographyon the supernate with ammonium sulfate, and carrying out ultrafiltration dialysis, thereby obtaining a purified HbcAg protein. The HBcAg obtained by using the method is high in yield, high in purity,simple in step and high in activity, and a high-specificity diagnosis raw material can be provided for an in-vitro diagnosis reagent.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for expressing and purifying HBcAg. Background technique [0002] Hepatitis B (hepatitis B) is caused by hepatitis B virus (hepatitis B virus, HBV), transmitted through blood and body fluids, mainly liver damage infectious disease. [0003] Hepatitis B core antigen (HbcAg) is encoded by the C region of the hepatitis B virus gene and translated from the second ATG. It is a polypeptide containing 183-185 amino acids and has a molecular weight of about 21KD. It is the only structural protein of the hepatitis B virus core particle. The C-terminus of HBcAg contains arginine and a variety of protease action sites, has the ability to bind nucleic acids, and is closely related to its assembly into hepatitis B core particles. During the assembly process of virus particles, HBcAg first forms homodimers, and the dimers further aggregate to form HBcAg particles. The real particl...

Claims

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Application Information

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IPC IPC(8): C07K14/02C07K1/36C07K1/34C07K1/18C12N15/81
CPCC07K14/005C12N15/815C12N2730/10122
Inventor 许德晨景宏维黄力
Owner GETEIN BIOTECH
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