ScFv antibody, encoding gene thereof and application of scFv antibody to preparation of preparation for treating or preventing hepatitis B

A coding and single-chain antibody technology, applied in the field of genetic engineering, can solve the problems of lack of effective preventive measures for emergency prevention of hepatitis B accidents, and achieve the effects of avoiding the spread of diseases, reducing costs, and increasing production

Active Publication Date: 2015-07-08
JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the Chinese health department banned the use of HBIG blood products in 1996, there is currently a lack of effective preventive measures for the prevention of hepatitis B after blood transfusion and the emergency prevention of accidents

Method used

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  • ScFv antibody, encoding gene thereof and application of scFv antibody to preparation of preparation for treating or preventing hepatitis B
  • ScFv antibody, encoding gene thereof and application of scFv antibody to preparation of preparation for treating or preventing hepatitis B
  • ScFv antibody, encoding gene thereof and application of scFv antibody to preparation of preparation for treating or preventing hepatitis B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Acquisition of anti-preS1scFv single-chain antibody encoding gene

[0073] 1. Initial sample

[0074] The peripheral blood leukocytes of blood donors with high titers of surface antigen antibodies were used as the initial samples as the source of antibody library genes. This can not only increase the kurtosis of natural antibodies, but also select neutralizing antibodies against popular strains, which can achieve multiplier effect with half the effort.

[0075] 2. RNA extraction

[0076] Take fresh peripheral blood leukocytes 10 6 Add 1 ml of cold TRIzol to a pre-cooled Eppendorf (Ep) tube, pipet repeatedly, mix well, place on ice for 10 min, centrifuge at 12000 rpm for 10 min at 4°C. Transfer the supernatant to a new Ep tube, add 200 μl of pre-cooled phenol-chloroform (phenol:chloroform=1:5, volume ratio), shake vigorously for 30 sec, centrifuge at 12000 rpm and 4°C for 10 min. The supernatant was taken, and 200 μl of phenol-chloroform was repeatedly add...

Embodiment 2

[0195] Example 2. Preparation of anti-preS1scFv single-chain antibody

[0196] 1. Construction of single-chain antibody expression vector (pET-27b-anti-preS1scFv)

[0197] The primer design software Primer Premier 5.0 was used to design PCR primers for the cloning of single chain antibody genes. When designing the upstream primers, Nco I restriction sites were added, and XhoI restriction sites were added downstream, which were synthesized by Invitrogen. The specific primer sequences are as follows:

[0198] anti-preS1scFv-F: CCATGG GT-TCGGTGCAGTTGGTG (the underlined part is the recognition sequence of Nco I, the sequence after - is the 1-15th position of sequence 2);

[0199] anti-preS1scFv-R: CTCGAG TTAACGTTTGATATCC (the underlined part is the recognition sequence of XhoI, the sequence after - is the reverse complement of the 753rd-768th position of sequence 2).

[0200] Using primers, the recombinant plasmid (carrying the DNA fragment shown in SEQ ID NO: 2 in the sequ...

Embodiment 3

[0210] Example 3. Performance detection of anti-preS1scFv single-chain antibody

[0211] 1. ELISA detects the affinity of anti-preS1 scFv antibody

[0212] The anti-preS1 scFv antibody prepared in Example 2 was coated on an ELISA plate overnight at 4°C, with a concentration gradient of 100 μg / mL, 50 μg / mL, 10 μg / mL, and 5 μg / mL, for the detection of anti-preS1 scFv on preS1 The specificity of the protein was also set as a negative control group. After coating overnight, wash 3 times with PBST, 2 min each time, block with 5% (5g / 100ml) nonfat milk powder at 37°C for 2h, after washing, add 10μg / mL preS1 protein (amino acid sequence is sequence 4 in the sequence table) , incubate at 37°C for 1 h, wash as before, add primary antibody (preS1 monoclonal antibody, product of Fitzgerald (Massachusetts, America), whose product catalog number is Cat.No.10-H07A; diluted 2000 times), incubate and wash as above. Then add the secondary antibody (HRP-goat anti-mouse antibody, a product of ...

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Abstract

The invention discloses an scFv antibody, an encoding gene thereof and application of the scFv antibody to preparation of a preparation for treating or preventing hepatitis B. The single-chain antibody is composed of a heavy-chain variable region, a light-chain variable region and a joining region between the heavy-chain variable region and the light-chain variable region, wherein the amino acid sequence of the heavy-chain variable region ranges from the first position to the 127th position counted from the N tail end of a sequence 1 in a sequence table; and the amino acid sequence of the light-chain variable region ranges from the 143rd position to the 255th position counted from the N tail end of a sequence 1 in the sequence table. Compared with a blood product, the single-chain antibody disclosed by the invention has the following advantages that no immune serum is needed, and the problem that the immune serum is limited because of few sources is solved; the risk that diseases are spread through blood because the blood product contains HBIG is avoided; the scFv antibody belongs to a human-derived protein so as not to generate immunological rejection reaction of an organism when being directly applied to a human body; and the scFv antibody is suitable for large-scale industrial production, the yield can be increased, and the cost can be reduced.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a scFv antibody, its coding gene and its application in preparing preparations for treating or preventing hepatitis B. Background technique [0002] Hepatitis B is a worldwide disease caused by hepatitis B virus (HBV). According to statistics, the number of people carrying hepatitis B surface antigen (HbsAg) in the world exceeds 280 million. my country is a highly prevalent area of ​​hepatitis B, 40-60% of the population has been infected with HBV, and about 120 million people carry the surface antigen of hepatitis B virus. Pregnant women who carry hepatitis B virus can pass the virus to their newborns. Moreover, the earlier life is infected with hepatitis B virus, the easier it is to form a persistent carrier, which can not only lead to chronic liver disease, but some of them can also develop into liver cirrhosis and liver cancer. [0003] There are two biological products for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/51G01N33/576A61K39/42A61P31/20C12N15/70C12N1/21C12R1/19
Inventor 李德山白银王文飞
Owner JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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