Humanized single chain antibody for anti-human cytomegalovirus envelope glycoprotein

Abstract

The present invention provides a human single strand antibody of resisting human cytomegalo virus envelope glycoprotein B characterized in that : it is expressed by protein first level sequence SEQ ID NO:1; it possesses character for identifying human cytomegalo virus envelope glycoprotein B and neutralizing activity for resisting human cytomegalo virus infection. The present invention also relates to nucleotide coding sequence corresponding to human single strand antibody sequence. The merit of the invention : The human single strand antibody for resisting human cytomegalo virus infection has not species difference to human, can not result in immune damage. The antibody is specific and has good binding ability and can serve as specific antibody drug for clinical anti-virus prophylaxis and treatment.

Description

technical field

[0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to a humanized single-chain antibody against envelope glycoprotein B of human cytomegalovirus. Background technique

[0002] Human cytomegalovirus (HCMV, human cytomegalovirus) is a DNA virus with the largest genome in the β-subfamily of the Herpesviridae family. It encodes more than 227 proteins with species specificity. In developed countries, the population infection rate is 40% to 60%, and in developing countries, the infection rate is even higher. In my country, the adult infection rate is 70% to 90%. HCMV infection can affect multiple organs, causing severe disease. HCMV infection is the most common intrauterine infection and the leading cause of congenital malformations, progressive deafness and mental retardation in newborns. There are about 40,000 children with congenital deformities caused by HCMV infection in my country every year. At the sam...

Examples

example 1

[0046] Example 1, clinical patient peripheral blood lymphocyte separation and RNA extraction

[0047] Take a 15ml centrifuge tube; add 8ml of lymphocyte separation solution to each tube. Gently superimpose 4ml of blood from each specimen on the separation medium; centrifuge at 3000rpm for 20 minutes; gently absorb the lymphocyte layer at the layered interface and centrifuge at 3000RPM for 20 minutes; gently suck off the supernatant, add 0.5ml Tripure and mix well. Extract RNA. The results of RNA extraction are shown in Figure 1

example 2

[0048] Example 2, Amplification of Humanized Single Chain Antibody Gene Fragment

[0049] The RNA of the sample collected in Example 1 was extracted, and the gene coding fragments of the antibody heavy chain and light chain variable regions were amplified by reverse transcription-polymerase chain reaction (RT-PCR), and the heavy chain and light chain variable regions were amplified. Assemble together by overlap-PCR. Among them, the reverse transcription reaction conditions: 30°C for 10 minutes; 42°C for 60 minutes; 99°C for 5 minutes; 4°C for 5 minutes. PCR conditions: 94°C for 5 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, 35 cycles; 72°C for 10 min; 4°C for 10 min. Overlap-PCR conditions: 94°C for 5 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 2 min, cycle 25 times; 72°C for 10 min; 4°C for 10 min. The result obtained is as Figure 2-Figure 5 shown.

example 3

[0050] Example 3, Construction of Antibody Library

[0051] The recovered and purified overlap-PCR product and the carrier plasmid pComb3X were respectively digested with SfiI, and the digested single-chain antibody fragment and vector were recovered and purified, ligated with DNA ligase, and incubated at room temperature for 4 hours to overnight. The connected samples and the corresponding number of electroporation cups were placed in an ice bath for 10 min. At the same time, take electrocompetent bacteria and dissolve them on ice. Mix the connected samples with competent bacteria and add them to the electroporation cup. Ice bath for 1 minute, the electroporator was set to 25μF, 2.5kV, 200Ω, and the duration was about 4-5 milliseconds. Rinse the electro-cup immediately with 1, 2, and 2ml of SOC medium in batches, mix the washing solution in a 50ml test tube, and shake at 37°C and 250rpm for 1 hour. Add 10ml preheated SB medium, 3ul 100mg / ml carbenzyl (if using XL1-blue, ad...

technical field

[0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to a humanized single-chain antibody against envelope glycoprotein B of human cytomegalovirus. Background technique

[0002] Human cytomegalovirus (HCMV, human cytomegalovirus) is a DNA virus with the largest genome in the β-subfamily of the Herpesviridae family. It encodes more than 227 proteins with species specificity. In developed countries, the population infection rate is 40% to 60%, and in developing countries, the infection rate is even higher. In my country, the adult infection rate is 70% to 90%. HCMV infection can affect multiple organs, causing severe disease. HCMV infection is the most common intrauterine infection and the leading cause of congenital malformations, progressive deafness and mental retardation in newborns. There are about 40,000 children with congenital deformities caused by HCMV infection in my country every year. At the sam...

Examples

example 1

[0046] Example 1, clinical patient peripheral blood lymphocyte separation and RNA extraction

[0047] Take a 15ml centrifuge tube; add 8ml of lymphocyte separation solution to each tube. Gently superimpose 4ml of blood from each specimen on the separation medium; centrifuge at 3000rpm for 20 minutes; gently absorb the lymphocyte layer at the layered interface and centrifuge at 3000RPM for 20 minutes; gently suck off the supernatant, add 0.5ml Tripure and mix well. Extract RNA. The results of RNA extraction are shown in Figure 1

example 2

[0048] Example 2, Amplification of Humanized Single Chain Antibody Gene Fragment

[0049] The RNA of the sample collected in Example 1 was extracted, and the gene coding fragments of the antibody heavy chain and light chain variable regions were amplified by reverse transcription-polymerase chain reaction (RT-PCR), and the heavy chain and light chain variable regions were amplified. Assemble together by overlap-PCR. Among them, the reverse transcription reaction conditions: 30°C for 10 minutes; 42°C for 60 minutes; 99°C for 5 minutes; 4°C for 5 minutes. PCR conditions: 94°C for 5 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, 35 cycles; 72°C for 10 min; 4°C for 10 min. Overlap-PCR conditions: 94°C for 5 min; 94°C for 30 sec, 56°C for 30 sec, 72°C for 2 min, cycle 25 times; 72°C for 10 min; 4°C for 10 min. The result obtained is as Figure 2-Figure 5 shown.

example 3

[0050] Example 3, Construction of Antibody Library

[0051] The recovered and purified overlap-PCR product and the carrier plasmid pComb3X were respectively digested with SfiI, and the digested single-chain antibody fragment and vector were recovered and purified, ligated with DNA ligase, and incubated at room temperature for 4 hours to overnight. The connected samples and the corresponding number of electroporation cups were placed in an ice bath for 10 min. At the same time, take electrocompetent bacteria and dissolve them on ice. Mix the connected samples with competent bacteria and add them to the electroporation cup. Ice bath for 1 minute, the electroporator was set to 25μF, 2.5kV, 200Ω, and the duration was about 4-5 milliseconds. Rinse the electro-cup immediately with 1, 2, and 2ml of SOC medium in batches, mix the washing solution in a 50ml test tube, and shake at 37°C and 250rpm for 1 hour. Add 10ml preheated SB medium, 3ul 100mg / ml carbenzyl (if using XL1-blue, ad...

Images