CN101220085B - Human cytomegalovirus recombinant protein and method for preparing the same - Google Patents

Abstract

The invention provides a human cytomegalovirus recombinant protein and the preparation method, relating to the field of genetic engineering technology, diagnostic reagent and vaccine development. The recombinant protein provided by the invention comprises subsequently 120 amino acids of the HCMV gp52 from the 261st to the 380th of the N-end, 54 amino acids of the pp150 from the 587th to the 640thof the N-end, 65 amino acids of the pp65 from the 361st to the 425th of the N-end, 44 amino acids of the gB from the 200th to the 243rd of the N-end, and 34 amino acids of the pp28 from the 95th to the 128th of the N-end from the N-end to the C-end. The human cytomegalovirus recombinant protein has a protein sequence with high specificity and strong immunogenicity that can improve the sensitivityand specificity of the test reagent, while the adoption of the multi antigenic determinant series recombinant protein technology can simplify the purification process and improve the working efficiency.

Description

Human cytomegalovirus recombinant protein and preparation method thereof

Technical field

The present invention relates to genetic engineering technique and diagnostic reagent, vaccine development field, particularly relate to a kind of human cytomegalovirus recombinant protein and preparation method thereof.

Background technology

Human cytomegalic inclusion disease virus (Human Cytomegalovirus is called for short HCMV) belongs to simplexvirus β subfamily, is a kind of dna virus, and it infects in the crowd and extensively exists, and cytomegalovirus infection is quite serious in the population of China.It is to cause that fetal in utero infects and grow damaged major reason that the women is subjected to the CMV primary infection at the gestation initial stage, in the fetus and newborn infant of pregnancy period primary infection HCMV, 80% can cause mental retardation, deformity and death [MiddeldorpJM.Jongsma J, Haar AT, et al.Detection of immunoglobulin M andG antibodies against cytomegalovirus early and late antigendby enzyme-linked immunosorbent assay.J Clin.Microbiol, 1984; 20 (4): 763-771.].HCMV also is one of organ transplantation failure and acquired immune deficiency syndrome (AIDS) patient accompanying infection main causes of death.Non-immune deficiency person is often caused long-term inapparent infection, even cause [Huang ES.Thepathogenicity of Human Cytomegalovirus.Springer-Vellag.Berin, 1993, the 1-45] such as conversion, distortion or cancerations of cells infected.Therefore diagnose the HCMV virus infection to have crucial meaning fast and accurately.

The cytomegalovirus infection diagnostic method has cast-off cells and tissue pathology checking at present, virus is separated, molecular hybridization test and Serological testing, but because the first three methods technical equipment requires height, the limitation that sense cycle is long, can't be extensive use of, and Serological testing has simple and rapid characteristic it is used widely clinically, the HCMV proteantigen that at present most detection reagent adopts box to use mainly is to derive from viral cultures or recombinant expressed single protein fragments, perhaps because proteantigen purity is low and poor specificity, causes false positive and reduce detection specificity; Perhaps because the contained antigenic determinant of single fragment antigen albumen is less, the antibody of being discerned is corresponding less, easily cause false negative and reduce detection sensitivity, though preparing detection reagent, multiple single fragment combination also can avoid the problems referred to above, but inevitable requirement is repeatedly extracted purge process, and the requirement of the purification techniques of small protein or polypeptide is higher, and working efficiency is too low.

In addition, in the world, lack under the situation of effectively treating virus infective medicament at present, become a kind of important medical intervention means by the vaccination prophylaxis of viral infections, the development of HCMV vaccine also is to carry out one of key areas of HCMV research, and an important prerequisite of vaccine research to be its used proteantigen should have high specific, strong immunogenicity and the complete antigenic determinant of trying one's best, it is very necessary therefore developing the placed in-line recombinant protein technology of a kind of multi-antigenic determinant.

Summary of the invention

In view of above problem, main purpose of the present invention is to provide a kind of human cytomegalovirus recombinant protein and preparation method thereof, this invention utilizes reorganization polymerase chain reaction (Polymerase Chain Reaction, abbreviation PCR) technique construction goes out to contain high degree of specificity and the placed in-line human cytomegalic inclusion disease virus of strong immunogenic multi-antigenic determinant (HumanCytomegalovirus is called for short HCMV) recombinant protein.

For reaching above purpose, human cytomegalovirus recombinant protein provided by the invention, it is shown in amino acid residue sequence SEQ ID No.1: described recombinant protein contain successively from the N-end to the C-end 54 amino acid of HCMV gp52 from 120 amino acid of terminal the 261st to the 380th of N-, pp150 from terminal the 587th to the 640th of N-, pp65 from 65 amino acid of terminal the 361st to 425 of N-, gB from terminal the 200th to 243 of N-44 amino acid and pp28 from 34 amino acid of terminal the 95th to 128 of N-.

Wherein the dna sequence dna of above-mentioned recombinant protein is shown in SEQ ID No.2, described gp52 purpose peptide segment DNA sequence is the 781st to the 1140th Nucleotide of gp52, pp150 purpose peptide segment DNA sequence is the 1759th to the 1920th Nucleotide, pp65 purpose peptide segment DNA sequence is the 1081st to the 1275th Nucleotide, gB purpose peptide segment DNA sequence is the 598th to the 729th Nucleotide, and pp28 purpose peptide segment DNA sequence is the 283rd to the 384th Nucleotide.

Human cytomegalovirus recombinant protein preparation method provided by the invention, its gene recombination and purge process may further comprise the steps:

(1) is designed for the PCR primer of amplification gp52 target DNA fragment to (p1, p2), be used to increase the PCR primer of pp150 target DNA fragment to (p3, p4), be used to increase the PCR primer of pp65 target DNA fragment to (p5, p6), be used to increase the PCR primer of gB target DNA fragment to (p7, p8) and the PCR primer of the pp28 target DNA fragment that is used to increase to (p9, p10), primer p1 and p10 have the restriction enzyme site of NdeI and XhoI respectively, primer p2 and p3 order are complementary, and jointly corresponding to gp52 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp150, primer p4 and p5 order are complementary, and jointly corresponding to pp150 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp65, primer p6 and p7 order are complementary, and jointly corresponding to pp65 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of gB, primer p8 and p9 order are complementary, and jointly corresponding to gB above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp28;

(2) with the viral cultures be template, with primer P1 and P2 amplification gp52 fragment, with primer P3 and P4 amplification pp150 fragment, with primer P5 and P6 amplification pp65 fragment, with primer P7 and P8 amplification gB fragment, with primer P9 and P10 amplification pp28 fragment; Gp52 fragment and the pp150 fragment that obtains with first round pcr amplification is template again, adds primer P1 and P4, amplification gp52 pp150 fragment, and the pp65 fragment and the gB fragment that obtain with first round pcr amplification are template, add primer P5 and P8, amplification pp65gB fragment; Taking turns gp52pp150 and the pp65gB fragment that pcr amplification obtains with second then is template, adds primer P1 and P8, amplification gp52pp150pp65gB; Last fragment gp52pp150pp65gB and the pp28 that obtains with amplification is template, adds primer P1 and P10, and amplification gp52pp150pp65gB pp28 obtains placed in-line goal gene recombinant fragment: gp52 pp150pp65gBpp28;

(3) with pET28a carrier and pcr amplification product through NdeI and XhoI double digestion, product purification is after T ₄Dna ligase connects, and connects product transformed into escherichia coli Top10 competence, coats on the LB flat board that contains kantlex, is inverted overnight incubation for 37 ℃, selects the bacterium colony of growth next day, carries out PCR and dna sequencing and identifies, obtains correct positive transformant;

(4) extract the positive transformant plasmid, change DE3 competence bacteria in the BL21 series over to chemical transformation, containing 37 ℃ of inversion overnight incubation on the LB flat board of kantlex, select the dull and stereotyped bacterium colony LB culture medium culturing that contains kantlex that goes up growth next day, induce the expression strain of LB cultivation with IPTG, gather in the crops thalline behind the abduction delivering, obtain the recombinant protein of purifying through carrying out ultrasonic bacteria breaking, Histidine affinity column and ion-exchange chromatography.

Human cytomegalovirus recombinant protein provided by the invention has high specific, strong immunogenic protein sequence, can improve the susceptibility and the specificity of detection reagent, the false negative that causes when avoiding adopting single fragment antigen protein and reduce the detection sensitivity problem, and this recombinant protein has been taked the placed in-line recombinant protein technology of multi-antigenic determinant, can simplify purification process, increase work efficiency.In addition, this recombinant protein provided by the invention also has good practical value in human cytomegalic inclusion disease virus vaccine development field.

The amino acid residue sequence of human cytomegalovirus recombinant protein of the present invention (SEQID No.1):

NH3-FAVENFLTEEPFQRGDPFDKNYVGNSGKSRG

GGGGGGSLSSLANAGGLHDDGPGLDNDLMNEPMG

LGGLGGGGGGGGKKHDRGGGGGSGTRKMSSGGGG

GDHDHGLSSKEKYEQHKITSYAGAGAAILTPTPV

NPSTAPAPAPTPTFAGTQTPVNGNSPWAPTAPLP

GDMNPANPQYSEHPTFTSQYRIQGKLEYRHTWDR

HDEGAAQGDDDVWTSGSDSDEELVTTERKTPRVT

GGGAAYHRDSYENKTMQLMPDDYSNTHSTRYVTV

KDQWHSRGSTWLYRRSTFREDKAPKPSKQSKKKK

KPSKHHHHQQSSIM-COOH

The dna sequence dna of human cytomegalovirus recombinant protein of the present invention (SEQ IDNo.2):

TTCGCCGTGGAGAATTTTCTCACCGAGGAACCTTTCCAGCGTGGCGATCCCTTCGACAA

AAATTACGTCGGGAACAGCGGCAAGTCGCGTGGCGGCGGCGGTGGTGGCGGCAGCCTCT

CTTCGCTGGCCAATGCCGGCGGTCTGCATGACGACGGCCCGGGTCTGGATAACGATCTC

ATGAACGAGCCCATGGGTCTCGGCGGTCTGGGAGGAGGTGGCGGCGGTGGCGGCAAGAA

GCACGACCGCGGTGGCGGCGGTGGTTCCGGTACGCGGAAAATGAGCAGCGGTGGCGGCG

GCGGTGATCACGACCACGGTCTTTCCTCCAAGGAAAAATACGAGCAGCACAAGATCACC

AGCTACGCTGGCGCCGGCGCTGCCATCCTCACGCCGACGCCTGTCAATCCTTCCACGGC

CCCCGCTCCGGCCCCGACACCTACCTTCGCGGGTACCCAAACCCCGGTCAACGGTAACT

CGCCCTGGGCTCCGACGGCGCCGTTGCCCGGGGATATGAACCCCGCCAACCCTCAGTAC

AGCGAGCACCCCACCTTCACCAGCCAGTATCGCATCCAGGGCAAGCTTGAGTACCGACA

CACCTGGGACCGGCACGACGAGGGTGCCGCCCAGGGCGACGACGACGTCTGGACCAGCG

GATCGGACTCCGACGAAGAACTCGTAACCACCGAGCGCAAGACGCCCCGCGTCACCGGC

GGCGGCGCCGCTTATCATAGGGACAGCTATGAAAACAAAACCATGCAATTAATGCCCGA

CGATTATTCCAACACCCACAGTACCCGTTACGTGACGGTCAAGGATCAATGGCACAGCC

GCGGCAGCACCTGGCTCTATCGTCGCTCGACGTTTCGCGAGGACAAGGCTCCGAAACCG

AGCAAGCAGTCAAAAAAGAAAAAGAAACCCTCAAAACATCACCACCATCAGCAAAGCTC

CATTATG

Embodiment

Below in conjunction with specific embodiment the present invention is described in further detail, but not as a limitation of the invention.

Human cytomegalovirus recombinant protein and preparation thereof

1. the preparation before the reorganization

According to the segmental dna sequence dna of purpose, promptly HCMV gp52, pp150, pp65, gB and pp28 go up the cDNA sequence of purpose peptide section and the restriction enzyme site on the plasmid, design five couples of PCR primer (P1, P2), (P3, P4), (P5, P6), (P7, P8) and (P9, P10).P1 and p2 the 781st to the 1140th Nucleotide of gp52 that is used to increase, p3 and p4 are used to increase the 1759th of pp150 to the 1920th Nucleotide, p5 and p6 are used to increase the 1081st of pp65 to the 1275th Nucleotide, p7 and p8 are used to increase the 598th of gB to the 729th Nucleotide, and p9 and p10 are used to increase the 283rd of pp28 to the 384th Nucleotide; Primer p1 and p10 have the restriction enzyme site of NdeI and XhoI respectively, primer p2 and p3 order are complementary, and jointly corresponding to gp52 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp150, primer p4 and p5 order are complementary, and jointly corresponding to pp150 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp65, primer p6 and p7 order are complementary, and jointly corresponding to pp65 above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of gB, primer p8 and p9 order are complementary, and jointly corresponding to gB above-mentioned segmental 3 ' the terminal and above-mentioned segmental 5 ' end sequence of pp28.

2. target DNA fragment PCR reorganization

First round pcr amplification: with the human cytomegalic inclusion disease virus culture is template, with primer P1 and P2 amplification gp52 target DNA fragment, with primer P3 and P4 amplification pp150 target DNA fragment, with primer P5 and P6 amplification pp65 target DNA fragment, with primer P7 and P8 amplification gB target DNA fragment, with primer P9 and P10 amplification pp28 target DNA fragment.

Second takes turns pcr amplification: the gp52 target DNA fragment and the pp150 target DNA fragment that obtain with first round pcr amplification are template, add primer P1 and P4, amplification gp52 pp150 target DNA fragment.The pp65 target DNA fragment and the gB target DNA fragment that obtain with first round pcr amplification are template, add primer P5 and P8, amplification pp65gB target DNA fragment.

The third round pcr amplification: taking turns gp52 pp150 and the pp65gB target DNA fragment that pcr amplification obtains with second is template, adds primer P1 and P8, amplification gp52 pp150pp65gB target DNA fragment.

The four-wheel pcr amplification: the pp28 target DNA fragment that gp52 pp150pp65gB target DNA fragment that obtains with third round amplification and first round amplification obtain is a template, adds primer P1 and P10, amplification gp52 pp150pp65gB pp28 target DNA fragment.Obtain complete target DNA recombinant fragment: gp52 pp150pp65gB pp28.

3. recombinant dna fragment inserts expression plasmid

PET28a carrier and pcr amplification product gp52 pp150pp65gB pp28 target DNA fragment are through NdeI and XhoI double digestion, and product purification is after T ₄Dna ligase is connected with carrier, connects product and is transformed into clone strain Top10, coats the LB flat board that contains kantlex and is inverted overnight incubation for last 37 ℃, selects the dull and stereotyped bacterium colony of going up growth next day and carries out PCR and dna sequencing evaluation recon.

4. at the expression in escherichia coli recombinant antigen protein

Correct transformant is cultivated back upgrading grain and is changed the DE3 bacterial strain of expressing in the BL21 series over to, coat the LB that contains penbritin and be inverted overnight incubation for dull and stereotyped last 37 ℃, select the dull and stereotyped bacterium colony LB culture medium culturing that contains kantlex that goes up growth next day, with IPTG abduction delivering 5 hours, collect thalline obtains purifying through carrying out ultrasonic bacteria breaking, Histidine affinity column and ion-exchange chromatography recombinant protein.

5. recombinant protein Western-blot checking

For verifying that reorganization HCMV protein and anti-HCMV antiserum(antisera) are reactive and the goal gene of recombinating obtains expression and has antigenicity, test with the Western-blot method.Positive serum is: 10 parts of rabbit anti-HCMV antibody positive serum and the 30 parts of anti-HCMV antibody positive of people serum (detecting confirmation through the ELISA method) with HCMV viral cultures immune rabbit results.Negative serum is: 10 parts contrast normal rabbit serum and the 30 parts of anti-HCMV negative antibody of people serum (detecting confirmation through the ELISA method).

Test result such as following table:

Serum sample	Total routine number	Positive routine number	Negative routine number	Positive rate	Negative recall rate
						The anti-HCMV antibody positive of rabbit serum	10	10	0	100％	0

Serum sample	Total routine number	Positive routine number	Negative routine number	Positive rate	Negative recall rate
						The contrast normal rabbit serum	10	0	10	0	100％
The anti-HCMV antibody positive of people serum	30	30	0	100％	0
						The anti-HCMV negative antibody of people serum	30	0	30	0	100％

The result shows, 10 parts of rabbit anti-HCMV antibody positive serum and the 30 parts of anti-HCMV antibody positive of people serum with HCMV viral cultures immune rabbit gained all produce positive reaction with antigen expressed, and 10 parts of contrast normal rabbit serums and 30 parts of people's anti-HCMV negative antibody serum and antigen expressed all produce negative reaction.Results suggest reorganization goal gene obtains to express, and reorganization HCMV protein and totivirus protein have tangible cross reactivity, and have very strong specificity, have the practical application meaning.

Claims (2)

1. a human cytomegalovirus recombinant protein is characterized in that, its amino acid residue sequence is shown in SEQ IDNo.1.

2. coding claim 1 described protein DNA, the nucleotide sequence that it is characterized in that described DNA is shown in SEQ IDNo.2.