CN104387457B - Neomorph mutant protein for encoding gene SLC10A1 of chronic hepatitis B virus and hepatitis D virus hepatocyte receptors as well as encoding gene and application of neomorph mutant protein and encoding gene - Google Patents

Abstract

The invention discloses a mutant strain of the coding gene SLC10A1 of liver cell receptor of hepatitis B virus and hepatitis D virus, mutant protein and its application in the prevention or treatment of hepatitis B virus and hepatitis D virus infection and related liver failure. The mutant protein of the SLC10A1 gene is mutated from serine to phenylalanine at position 267 of the amino acid sequence of the SLC10A1 protein. According to the mutant protein and its coding gene, a diagnostic kit based on the SLC10A1 gene and its mutant can be established. And because this mutation prevents hepatitis B virus or hepatitis D virus from entering liver cells, it has the prospect of producing corresponding new drugs for the prevention and treatment of hepatitis B or hepatitis D. This study also suggests that other mutations in the SLC10A1 gene may also have a role in the prevention and treatment of hepatitis B and the application prospect of DG.

Description

New expression of the gene SLC10A1 encoding the hepatocyte receptor of chronic hepatitis B and D virus Mutant proteins, coding genes and their applications

technical field

The invention relates to the fields of bioengineering and medical technology, in particular to a new mutant protein of the chronic hepatitis B and D virus liver cell receptor coding gene SLC10A1, the coding gene and application thereof.

Background technique

Chronic hepatitis B is an infectious disease caused by hepatitis B virus (HBV) infection with liver damage as the main manifestation, and it is a major disease related to public health in my country and even the world. The hepatitis B virus enters the liver cells through the hepatitis B virus liver cell receptor to cause infection, so the mutation of the receptor will interfere with the binding of the hepatitis B virus to it, thereby preventing the hepatitis B virus from entering the liver cells. Moreover, hepatitis D virus and hepatitis B virus share the same liver cell receptor, so the mutation of chronic hepatitis B virus liver cell receptor also has the effect of preventing hepatitis D virus from entering liver cells.

Contents of the invention

The object of the present invention is to provide the mutant strain of chronic hepatitis B and hepatitis D virus liver cell receptor coding gene SLC10A1 and the application of mutant protein in the prevention and treatment of hepatitis B, hepatitis D and related liver failure.

The content claimed in the present invention is based on the research basis of the mutant gene of the coding gene SLC10A1 of the liver cell receptor of chronic hepatitis B and D virus and the mutant protein encoded by it. Any new drugs and methods resulting from their application in the prevention and treatment of related liver failure

A mutant protein of the coding gene SLC10A1 of chronic hepatitis B and D virus hepatocyte receptor, which is composed of serine at the 267th amino acid sequence of the coding gene SLC10A1 protein of chronic hepatitis B and D virus hepatocyte receptor Mutation to phenylalanine, represented by p.Ser267Phe in the present invention. The amino acid sequence of the mutant protein of the gene SLC10A1 encoding the hepatocyte receptor of chronic hepatitis B and D virus is shown in SEQ ID NO:1.

The gene encoding the mutant protein of the chronic hepatitis B and D virus hepatocyte receptor encoding gene SLC10A1, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:2.

Application of the mutated protein of the coding gene SLC10A1 of chronic hepatitis B and hepatitis D virus liver cell receptor in the preparation of detection reagents for chronic hepatitis B virus.

Application of the coding gene of the mutant protein coding gene SLC10A1 of the chronic hepatitis B and hepatitis D virus hepatic cell receptor in the preparation of detection reagents for chronic hepatitis B virus.

The detection reagent scheme for preparing chronic hepatitis B virus hepatitis by the mutant protein of the coding gene SLC10A1 of the coding gene SLC10A1 of chronic hepatitis B and hepatitis D virus hepatocyte receptor and the coding gene of the mutant protein is as follows:

A detection kit for chronic hepatitis B or D virus, consisting of the following components: primers (the nucleotide sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:4), PCR reaction solution, PCR product purification Cassette and sequencing reaction solution. The PCR reaction solution is dissolved in Tag DNA polymerase, dNTPs, magnesium ions (such as MgCl ₂ ), and PCR reaction buffer (such as 10×Taq Buffer). The above reagents can be directly purchased from corresponding commodities, such as (10mM) dNTPs were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.; Tag DNA polymerase and its supporting 10×Taq Buffer, MgCl ₂ were purchased from Fermentas. The dosage and concentration of each reagent are conventional values in the field, such as 1u/μL Tag DNA polymerase 1μL, 2mmol/L dNTP (that is, a mixture of 4 dNTPs with a concentration of 2mmol/L) 3μL, 25mmol/L MgCl ₂ 3 μL, 10×Taq Buffer (containing (NH4) ₂ SO ₄ ) 3 μL, sterilized ddH ₂ O 17 μL.

The PCR product purification box is a solution for purifying PCR products and a DNA adsorption column Labell;

The solution of the purified PCR product is: 1u/μL shrimp alkaline phosphatase (SAP, available from Fermentas Company) 0.6 μL, 20u/μL exonuclease (Exo I, available from Fermentas Company) 0.15 μL and sterilized ddH ₂ O 1.25-1.75μL, or other volumes of mixed solution prepared according to the above ratio. The solution of purified PCR product is preferably 2-2.5 μL.

The sequencing reaction solution is a mixture of BigDye (purchased from ABI Company) and 5×Sequence buffer (purchased from ABI Company), the volume ratio of BigDye and 5×Sequence buffer is preferably 1:2, such as 1 μL BigDye and 2 μL 5×Sequence buffer mix. In the above chronic hepatitis B detection kit, the preferred primer nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the primer concentration is 3.2 μmol/L.

The present invention conducts whole exome sequencing on 99 chronic hepatitis B patients with "relatively extreme traits" and 90 normal controls who have not been injected with hepatitis B vaccine, and finds the most likely candidates through bioinformatics analysis and further data screening Gene; then in the case-control study with a large sample size of the candidate genes found (including 1899 cases of chronic hepatitis B patients, 1828 cases of normal controls without hepatitis B vaccine injection) to confirm the role of chronic hepatitis B virus hepatocyte receptors The mutation p.Ser267Phe of the coding gene SLC10A1 and its coded mutant protein can resist chronic hepatitis B and liver failure. At the same time, our structural analysis of the mutant protein also confirmed that this mutation interferes with the combination of hepatitis B virus and its receptors in liver cells, thereby preventing hepatitis B virus from entering liver cells.

The full-length nucleotide sequence of the human SLC10A1 gene (including the mutation site) of the present invention is usually carried out by polymerase chain reaction (PCR). For the PCR amplification method, primers can be designed according to the nucleotide sequence of the SLC10A1 gene, and the DNA extracted from the detected chronic hepatitis B patient can be used as a template to amplify the target sequence. Finally, the mutations of related genes were detected and confirmed by sequencing reaction.

Compared with prior studies, the present invention has the following beneficial effects:

The present invention first proves in clinical cases that the SLC10A1 variant strain and its coded mutant protein have the effect of resisting chronic hepatitis B and related liver failure. Because this mutation prevents hepatitis B virus from entering liver cells, it has the prospect of producing corresponding new drugs and methods for the prevention and treatment of hepatitis B, and because hepatitis B virus and hepatitis D virus share the same liver cell receptor, this discovery is also applicable Development and application of drugs and other therapies for the prevention and treatment of hepatitis D virus infection. This study also suggests that other mutations in the SLC10A1 gene may also have application prospects in the prevention and treatment of hepatitis B and hepatitis D.

Description of drawings

Figure 1 shows the partial sequencing results of the SLC10A1 gene, in which the upper part is the sequencing result of the wild type SLC10A1, the middle part is the sequencing result of the heterozygous mutation of SLC10A1p.Ser267Phe, and the lower part is the sequencing result of the homozygous mutation of SLC10A1p.Ser267Phe; the arrow shows mutation site.

Fig. 2 is a schematic diagram of the overall structure of the SLC10A1 p.Ser267Phe mutant protein.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1

1. Case collection: 1899 patients with chronic hepatitis B from the Department of Infectious Diseases of the Third Affiliated Hospital of Sun Yat-sen University and 1828 healthy volunteers who had not been vaccinated against hepatitis B or had unknown self-reported vaccination history from the Second Affiliated Hospital of Sun Yat-sen University After obtaining the informed consent of the above-mentioned patients, volunteers and family members, peripheral blood was collected and stored in EDTA anticoagulant tubes for future use. DNA was extracted from anticoagulated peripheral blood with QIAamp DNABlood Mini Kits (kit) produced by Qiagen (refer to the product manual for specific extraction steps), dissolved in TE, and stored at -80°C for later use.

2. Mutation detection of SLC10A1 gene:

The mRNA sequence of the SLC10A1 gene was obtained from Genbank (NM_003049). Genomic DNA sequences were obtained from UCSC database (http://genome.ucsc.edu/) and Ensembl database (http://asia.ensembl.org/index.html).

The detection steps are as follows:

1. PCR amplification:

The PCR reaction volume is 30 μL: 10×Taq Buffer (containing (NH4) ₂ SO ₄ ) 3 μL, 25 mmol/L MgCl ₂ 3 μL, 2 mmol/L dNTP mixture 3 μL, 3.2 μmol/L upstream and downstream primers 1 μL each, 1u/μL 1 μL of Taq DNA polymerase, 1 μL of the DNA template extracted in Example 1, and sterilized ddH ₂ O to make up the volume to 30 μL.

PCR reaction conditions: 95°C pre-denaturation for 3min; 95°C for 30s, primer-specific annealing temperature for 1min, 72°C for 45s, 36 cycles; final extension at 72°C for 10min.

The sequences of the upstream primer P1 and downstream primer P2 for detecting the mutation site of the SLC10A1 gene are:

P1: 5'-CCATCTGCTGCGAAACTC-3';

P2: 5'-GGGCTACCTGGTTCTTAGTGA-3';

2. Sequencing reaction scheme:

①Pre-reaction: total reaction volume is 3 μL: 1u/μL shrimp alkaline phosphatase (SAP) 0.6 μL, 20u/μL exonuclease (Exo I) 0.1 (Fermentas company), PCR product 0.5-1.0 μL, sterilized ddH ₂ O to make up the volume to 3 μL. The reaction mixture was reacted on a PCR instrument, incubated at 37°C for 15 minutes, and then at 85°C for 15 minutes to inactivate the enzyme. After the reaction is completed, the reaction mixture is directly used as a template for the sequencing reaction.

② Sequencing reaction: ABI company's 96-well plate was used to carry out on GeneAmp 9700 PCR machine. The total reaction volume is 10 μL: 3 μL of pre-reaction product, 2 μL of 5×Sequence buffer (ABI company), 1 μL of BigDye (ABI company), 1 μL of primer (3.2 μmol/L), 3 μL of sterilized ddH ₂ O. Reaction conditions: 98°C for 2min; 96°C for 10s, 50°C for 5s, 60°C for 4min, 30 cycles; finally store at 4°C.

③Sequencing purification: directly on a 96-well plate.

a) After the sequencing reaction is completed, prepare 240 μL of anhydrous ethanol 6mL+3mol/L sodium acetate (pH5.2) mixed solution (that is, absolute ethanol: sodium acetate volume ratio = 25:1), and add 50 μL of mixed solution to each sample . Cover with foil and vortex to mix.

b) Store the sequencing product at -20°C in the dark for more than 20 minutes.

c) Centrifuge at 3700 rpm for 30 minutes at 20°C.

d) Pour off the supernatant, put it upside down on four layers of absorbent paper, and centrifuge at 360rpm for 10s.

e) Prepare 75% ethanol (the ratio of absolute ethanol 7.5mL+ddH ₂ O 2.5mL mixed solution), add 90μL mixed solution to each sample. Cover with foil and vortex to mix.

f) Store the sequencing product at -20°C in the dark for more than 20 minutes.

g) Centrifuge at 3700 rpm for 30 minutes at 20°C.

h) Pour off the supernatant, put it upside down on four layers of absorbent paper, and centrifuge at 360rpm for 10s.

i) In a ventilated place at room temperature, dry in the dark for more than 30 minutes.

j) Add 10 μL of ddH ₂ O to each sample, seal with foil paper, shake and centrifuge.

k) The sample to be tested is placed at 4°C in the dark for more than 1.5 hours to fully dissolve, and then sequenced on the ABI3730 sequencer.

3. Analysis of sequencing results: analysis with ABI sequence analysis software: the inventors analyzed 1899 patients with chronic hepatitis B from the Department of Infectious Diseases of the Third Affiliated Hospital of Sun Yat-sen University and 1828 patients from the Second Affiliated Hospital of Sun Yat-sen University who had not been injected with hepatitis B vaccine or Healthy volunteers with unknown self-reported vaccination history were tested for the mutation of SLC10A1 gene.

The results are as follows: mutation site: the 267th position of the amino acid sequence of the SLC10A1 gene is mutated from serine to phenylalanine, which can also be expressed as p.Ser267Phe, and the amino acid sequence after mutation is shown in SEQ ID NO:1. Among them, 149 patients and 348 normal controls had heterozygous mutations. Homozygous mutations existed in 5 patients and 26 normal controls. In 1899 cases and 1828 controls, the number of risk alleles was 159 and 400, respectively, and the odds ratio (OR value) was 0.36. The P value is 5.7×10 ⁻²³ . In the study of polygenic genetic diseases, the risk genes are usually determined by association analysis. In the results of this experiment, the P value has a very significant difference, and the OR value is 0.36, indicating that the mutant gene has resistance to chronic viral hepatitis B and The role of associated liver failure. The sequencing results are shown in Figure 1.

Example 2

SLC10A1p.Ser267Phe mutant protein structure prediction: use the program PyMol (http://pymol.org), Discovery Studio 3.0 (Accelrys Inc.), the programs MultAlin (CorpetF. Multiple sequence alignment with hierarchical clustering. Nucleic. Acids.Res . 1988; 16, 10881-10890) and ESPript software (Gouet P, Robert X, Courcelle E.ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic. Acids. Res. 2003; 31, 3320- 3323) Predict the structure and functional domains of SLC10A1 p.Ser267Phe mutant protein.

The results of protein structure prediction are shown in Figure 2. The prediction of the protein structure domain encoded by the SLC10A1 gene, the protein includes two domains: the core region and the panel region, and the relative movement between the two domains forms an inward-open or outward-open structure. This suggests that the rigid body rotation of the core domain may facilitate the transport of bile acids. In the outward-open structure of our wild-type NTCP protein, we found that the 267th amino acid is located in the transmembrane domain 9b (TM9b), and specifically located in the ligand The surface of the migration pathway. At the same time, this model also shows that in the NTCP protein, amino acid 267 is very close to the necessary structural domain of hepatitis B virus infection composed of amino acid residues 157-165. In addition, the mutation of the hydrophilic serine to the large hydrophobic phenylalanine may affect the binding of NTCP to the ligand.

Example 3

Preparation of SLC10A1 gene mutation detection kit.

1. The components of the kit are shown in Table 1.

Table 1 Reagent name Specific ingredients Upstream primer P1 Dry powder 2OD, concentration after dissolution: 3.2μmol/L, volume: 1μL downstream primer P2 Dry powder 2OD, concentration after dissolution: 3.2μmol/L, volume: 1μL PCR reaction solution 1u/μL TaqDNA polymerase 1μL (purchased from Fermentas), 2mmol/LdNTP 3μL, 25mmol/LMgCl 23μL (TaqDNA polymerase matching reagent), 10×TaqBuffer (containing (NH ₄ ) ₂ SO ₄ , TaqDNA polymerase matching reagent) 3μL, Sterilize ddH2O 17 μL. PCR product purification kit 2-2.5 μL solution of purified PCR products (containing 1u/μL shrimp alkaline phosphatase (SAP, purchased from Fermentas company) 0.6 μL, 20u/μL exonuclease (Exo I, purchased from Fermentas company) 0.15 μL and inactivated bacteria ddH ₂ O1.25-1.75μL), DNA adsorption column Label Sequencing reaction solution Containing 1 μL BigDye and 2 μL 5×Sequencebuffer (both purchased from ABI Company)

2. The method of using the above kit: extract 2mL of blood from the patient to be tested, and use conventional methods (or a specific DNA extraction kit) to extract DNA from the blood. Dilute the PCR primers in the SLC10A1 Gene Mutation Detection Kit to 3.2 μmol/L, and use the extracted DNA as a template to perform a PCR reaction with the provided primers. Purify the PCR product using detection reagents and the provided PCR product purification kit. The purified product was sequenced directly after the sequencing reaction. Observe whether there is a missense mutation in the sequence obtained by sequencing, that is, whether there is a mutation at the 267th serine (Ser) in the amino acid sequence shown in SEQ ID NO:1.

SEQUENCE LISTING

<110> Sun Yat-sen University

<120> New mutant protein of chronic hepatitis B and D virus hepatocyte receptor encoding gene SLC10A1, encoding

Genes and their applications

<130>

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 349

<212> PRT

<213> A novel mutant protein of SLC10A1

<400> 1

Met Glu Ala His Asn Ala Ser Ala Pro Phe Asn Phe Thr Leu Pro Pro

1 5 10 15

Asn Phe Gly Lys Arg Pro Thr Asp Leu Ala Leu Ser Val Ile Leu Val

20 25 30

Phe Met Leu Phe Phe Ile Met Leu Ser Leu Gly Cys Thr Met Glu Phe

35 40 45

Ser Lys Ile Lys Ala His Leu Trp Lys Pro Lys Gly Leu Ala Ile Ala

50 55 60

Leu Val Ala Gln Tyr Gly Ile Met Pro Leu Thr Ala Phe Val Leu Gly

65 70 75 80

Lys Val Phe Arg Leu Lys Asn Ile Glu Ala Leu Ala Ile Leu Val Cys

85 90 95

Gly Cys Ser Pro Gly Gly Asn Leu Ser Asn Val Phe Ser Leu Ala Met

100 105 110

Lys Gly Asp Met Asn Leu Ser Ile Val Met Thr Thr Cys Ser Thr Phe

115 120 125

Cys Ala Leu Gly Met Met Pro Leu Leu Leu Tyr Ile Tyr Ser Arg Gly

130 135 140

Ile Tyr Asp Gly Asp Leu Lys Asp Lys Val Pro Tyr Lys Gly Ile Val

145 150 155 160

Ile Ser Leu Val Leu Val Leu Ile Pro Cys Thr Ile Gly Ile Val Leu

165 170 175

Lys Ser Lys Arg Pro Gln Tyr Met Arg Tyr Val Ile Lys Gly Gly Met

180 185 190

Ile Ile Ile Leu Leu Cys Ser Val Ala Val Thr Val Leu Ser Ala Ile

195 200 205

Asn Val Gly Lys Ser Ile Met Phe Ala Met Thr Pro Leu Leu Ile Ala

210 215 220

Thr Ser Ser Leu Met Pro Phe Ile Gly Phe Leu Leu Gly Tyr Val Leu

225 230 235 240

Ser Ala Leu Phe Cys Leu Asn Gly Arg Cys Arg Arg Thr Val Ser Met

245 250 255

Glu Thr Gly Cys Gln Asn Val Gln Leu Cys Phe Thr Ile Leu Asn Val

260 265 270

Ala Phe Pro Pro Glu Val Ile Gly Pro Leu Phe Phe Phe Pro Leu Leu

275 280 285

Tyr Met Ile Phe Gln Leu Gly Glu Gly Leu Leu Leu Ile Ala Ile Phe

290 295 300

Trp Cys Tyr Glu Lys Phe Lys Thr Pro Lys Asp Lys Thr Lys Met Ile

305 310 315 320

Tyr Thr Ala Ala Thr Thr Glu Glu Thr Ile Pro Gly Ala Leu Gly Asn

325 330 335

Gly Thr Tyr Lys Gly Glu Asp Cys Ser Pro Cys Thr Ala

340 345

<210> 2

<211> 1050

<212>DNA

<213> Coding sequence of SLC10A1 mutein

<400> 2

atggaggccc acaacgcgtc tgccccattc aacttcaccc tgccacccaa ctttggcaag 60

cgccccacag acctggcact gagcgtcatc ctggtgttca tgttgttctt catcatgctc 120

tcgctgggct gcaccatgga gttcagcaag atcaaggctc acttatggaa gcctaaaggg 180

ctggccatcg ccctggtggc acagtatggc atcatgcccc tcacggcctt tgtgctgggc 240

aaggtcttcc ggctgaagaa cattgaggca ctggccatct tggtctgtgg ctgctcacct 300

ggagggaacc tgtccaatgt cttcagtctg gccatgaagg gggacatgaa cctcagcatt 360

gtgatgacca cctgctccac cttctgtgcc cttggcatga tgcctctcct cctgtacatc 420

tactccaggg ggatctatga tggggacctg aaggacaagg tgccctataa aggcatcgtg 480

atatcactgg tcctggttct cattccttgc accataggga tcgtcctcaa atccaaacgg 540

ccacaataca tgcgctatgt catcaaggga gggatgatca tcattctctt gtgcagtgtg 600

gccgtcacag ttctctctgc catcaatgtg gggaagagca tcatgtttgc catgacacca 660

ctcttgattg ccacctcctc cctgatgcct ttattggct ttctgctggg ttatgttctc 720

tctgctctct tctgcctcaa tggacggtgc agacgcactg tcagcatgga gactggatgc 780

caaaatgtcc aactctgttt caccatcctc aatgtggcct ttccacctga agtcattgga 840

ccacttttct tctttcccct cctctacatg attttccagc ttggagaagg gcttctccctc 900

attgccatat tttggtgcta tgagaaattc aagactccca aggataaaac aaaaatgatc 960

tacacagctg ccacaactga agaaacaatt ccaggagctc tgggaaatgg cacctacaaa 1020

ggggaggact gctcccccttg cacagcctag 1050

<210> 3

<211> 18

<212>DNA

<213> Upstream primer P1

<400> 3

ccatctgctg cgaaactc 18

<210> 4

<211> 21

<212>DNA

<213> downstream primer P2

<400> 4

gggctacctg gttcttagtg a 21

Claims (2)

1. A new mutant protein of the coding gene SLC10A1 of chronic hepatitis B and hepatitis D virus hepatocyte receptor, characterized in that the amino acid sequence of the chronic hepatitis B and D virus hepatocyte receptor gene SLC10A1 protein is 267th The position is mutated from serine to phenylalanine; the amino acid sequence of the new mutant protein of the chronic hepatitis B and D virus hepatocyte receptor coding gene SLC10A1 is shown in SEQ ID NO:1. 2. The gene encoding the new mutant protein of the encoding gene SLC10A1 of the chronic hepatitis B and hepatitis D virus hepatocyte receptor described in claim 1, characterized in that its nucleotide sequence is as shown in SEQ ID NO:2.