Patent Application
20150284690
1. Field of Invention
The present invention relates to the field of biotechnology, in particular to a new hepatitis B virus mutation, mutant amplification kit and use thereof.
2. Description of Related Arts
By the current global estimates, there are 350 million chronic hepatitis B virus (HBV) carriers, and one-third of the global population infected with HBV. In China, Hepatitis B is one of the most serious, and most extensive infectious diseases, with the infection rate as high as 60%, and about 120 million people carry HBV. The incidence of hepatitis B ranks first in infectious diseases, and the mortality ranks third in infectious disease.
According to the different clinical manifestations of HBV infection, it can be divided into several types: asymptomatic carrier state, acute self-limited hepatitis, chronic hepatitis, fulminant hepatitis (FH). Mother-to-child transmission is a major route of transmission of HBV, the patient is usually in a state of immune tolerance, and the response to HBV does not occur, but most asymptomatic carriers tend to occur hepatitis, and even some eventually grows into FH. The mechanism of Hepatitis B aggravation has not been cleared by now, and is currently considered on account of two aspect, which are function of host immune and function of virus. The occurrence of HBV variation and severe hepatitis remains controversial, and there is no one gene variant can be acted as a symbol variant of severe hepatitis B. Moreover, a large number of studies have shown that HBV exists in the form of quasispecies in the patient's body, and the quasispecies characteristics of HBV should be taken into account as studying the relationship between virus mutation and hepatitis. Currently, most studies investigate HBV quasispecies by PCR-cloning-sequencing method. Therefore, it would offer help for the clinical early diagnosis and intervention of HBV, to find gene loci with closely related to hepatitis aggravation.
The object of the present invention is to provide a hepatitis B virus mutant with closely related to hepatitis aggravation, a mutant amplification kit, and its application, to offer help for the clinical early diagnosis and intervention of HBV.
One aspect of the invention discloses a hepatitis B virus (referred to as hepatitis B virus or HBV) mutant, including hepatitis B virus genome, a mutation of the hepatitis B virus mutant occurs in genome sequence No. 216 and/or 285 of the hepatitis B virus, the mutation occurred at position 216 is that base T mutates to C (216T→C), the mutation occurred at position 285 is that base G mutates to A (285G→A).
Preferably, the nucleotide sequence of the hepatitis B virus genome is selected from SEQ ID NO:1, SEQ ID NO:10 or SEQ ID NO: 11.
Further, in the complementary sequence of the HBV genome sequence of the hepatitis B virus mutant, the mutation occurred at the corresponding genomic locus to genome No. 216 is that A mutates to G, and the mutation occurred at the corresponding genomic locus to genome No. 285 is that C mutates to T.
Preferably, compare to the normal S protein of hepatitis B virus, the S protein encoded by the hepatitis B virus mutant features that its amino acid sequence occurs mutation at position 21 and/or 44, the amino acid mutation at position 21 is mutating from leucine (L) to serine (S), and the amino acid mutation at position 44 is mutating from glycine (G) to glutamic acid (E).
More preferably, the amino acid sequence of the normal S protein of the hepatitis B virus is SEQ ID NO: 12.
MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNS QSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTS TTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWL SLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI (SEQ ID NO:12)
The second aspect of the present invention discloses a HBV mutant amplification kit for amplifying the gene fragments of the hepatitis B virus genome sequence No. 216 and No. 285, including primers, dNTP, PCR buffer, DNA polymerization enzyme, and ddH2O, the primers includes an upstream primer and a downstream primer, the primer specific amplifying the gene fragments including the hepatitis B virus genome sequence No. 216 and No. 285.
Preferably, the DNA polymerization enzyme is a high fidelity DNA polymerization enzyme; more preferably, the high fidelity DNA polymerization enzyme is Prime Star.
Preferably, PCR buffer solution is 5× Prime Star buffer.
Preferably, the sequences of the upstream primer and downstream primer are as follows:
The PCR system of the kit of the present invention is as follows:
Dilute with ddH2O to the total volume of 50 μL.
Preferably, the kit of the present invention may also include DNA extraction reagent.
Preferably, the amplification procedure of the HBV mutant amplification kit of the invention is: predenaturation at 94° C. 3 min, then 94° C. 30 s, 57° C. 15 s, 72° C. 2 min for 45 cycles, extending at 72° C. for 10 min.
The third aspect of the present invention discloses a usage for the HBV mutant amplification kit in the following steps:
1) Preparation of DNA template: drawing the peripheral venous blood and extracting HBV genome as the DNA template;
2) PCR amplification: performing PCR amplification reaction of the DNA template as prepared in step 1) according to the above amplification procedure by the kit of the present invention;
3) Purifying PCR product: PCR product is recycled and purified via agarose gel electrophoresis.
Preferably, the amplification procedure in step 2) is: predenaturation at 94° C. 3 min, then 94° C. 30 s, 57° C. 15 s, 72° C. 2 min for 45 cycles, extending at 72° C. for 10 min.
Preferably, the concentration of the agarose gel electrophores used in step 2) is 1%.
The purified PCR product obtained by step 3) is sequenced by sanger sequencing technology or solexa sequencing technology, the sequencing results are compared with the sequence of SEQ ID NO: 1, SEQ ID NO: 10 or SEQ ID NO: 11, to confirm that whether the gene fragments of the hepatitis B virus genome sequence No. 216 and No. 285 corresponding to the PCR product exists the mutation of the present invention.
The fourth aspect of the present invention discloses a use of hepatitis B virus mutant or HBV mutant amplification kit in the preparation of a medicament for the clinical early diagnosis and prevention of the severe hepatitis B disease.
The fifth aspect of the present invention further discloses a method for clinical early diagnosis and prevention of the severe hepatitis B disease, to detect whether the subject blood has the hepatitis B virus mutant of the present invention.
Preferably, in the method for the clinical early diagnosis and prevention of the severe hepatitis B disease, the steps to detect whether the subject blood has the hepatitis B virus mutant are as follows:
1) Preparation of DNA template: drawing the peripheral venous blood of the test subject and extracting HBV genome as the DNA template;
2) PCR amplification: adding the DNA template as prepared in step 1) into the PCR reaction system, amplifying the gene fragments including the hepatitis B virus genome sequence No. 216 and No. 285;
3) Purifying PCR product: PCR product is recycled and purified via agarose gel electrophoresis;
4) Sequencing of the PCR product: the PCR product by purified and obtained by step 3) is sequenced by the gene sequencing technology;
5) Result analysis: the sequencing results of step 4) are compared with the sequence of SEQ ID NO: 1, SEQ ID NO: 10 or SEQ ID NO: 11, to confirm that whether the hepatitis B virus genome sequence No. 216 and No. 285 corresponding to the PCR product exists mutation.
More preferably, the PCR reaction system in the step 2) includes primers, dNTP, PCR buffer, DNA polymerization enzyme, and ddH2O, the primers includes an upstream primer and a downstream primer, amplifying the gene fragments including the hepatitis B virus genome sequence No. 216 and No. 285.
More preferably, the primer sequence is SEQ ID NO: 8-9.
More preferably, the DNA polymerization enzyme is a high fidelity DNA polymerization enzyme; optimally, the high fidelity DNA polymerization enzyme is Prime Star.
More preferably, PCR buffer solution is 5× Prime Star buffer.
More preferably, the amplification procedure in step 2) is: predenaturation at 94° C. 3 min, then 94° C. 30 s, 57° C. 15 s, 72° C. 2 min for 45 cycles, extending at 72° C. for 10 min.
More preferably, the gene sequencing technology in step 4) is sanger sequencing technology or solexa sequencing technology.
More preferably, the mutation occurred at position 216 of the hepatitis B virus genome sequence corresponding to the PCR product is that base T mutates to C, the mutation occurred at position 285 of the hepatitis B virus genomic sequence corresponding to the PCR product is that base G mutates to A.
The test subject that contains the mutant of the present invention in the blood tends to occur serious inflammation of the liver.
The mutation of NO. 216 (216T→C) and the mutation of NO. 285 (285G→A) of the hepatitis B virus genome sequence are closely related to the inflammation aggravation of the hepatitis B.
Since HBV in patient's body is in the form of quasispecies, and its genotype has a comparatively high mutation rate in humans, therefore, the present invention adopts the solexa high-throughput sequencing technology to detect complete HBV genome for screening out the loci closely associated with the hepatitis aggravation; and provides a HBV mutant amplification kit to perform amplification on the HBV genome carried by the hepatitis B patient via the designed specific primers, and to compare the sequences of the amplification products with the sequence of SEQ ID NO:1, SEQ ID NO:10 or SEQ ID NO:11 via gene sequencing method to detect whether T→C mutation at position 216 and/or G→A mutation at position 285 exists in the sample to be detected, thereby offering help for the clinical early diagnosis and intervention of severe hepatitis B, and providing references for further study on the functional changes caused by HBV gene mutation.
The present invention is further illustrated by combining with the specific embodiments. It should be understood that the following embodiments are merely used to explain the invention, but are not used to limit the scope of the invention.
First: serum Collection from Patients with Severe Hepatitis B and Chronic Hepatitis B
Collect the serum samples of 12 cases with severe hepatitis B and 12 cases with chronic mild hepatitis B from the first affiliated hospital of medical school of Zhejiang university (the patients has been diagnosed explicitly with severe or chronic mild hepatitis B by the indicators of ALT, AST, TBIL, PT, viral titer, etc., and clinical manifestations)
We download 616 complete sequences of HBV genome, and design three pairs of primers (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7) by using primer 5.0 software according to the complete genome conserved sequence of HBV.
An equal volume of nucleic acid extract is added into the 50 ul serum and mixed, followed by water bath at 100 for 10 min, centrifugation for 10 min with 13000 r/min, and then the supernatant is drawn for PCR amplification.
The amplification for the HBV gene of a patient adopts Prime Star fidelity enzymes of TaKaRa Company, with the brief steps as follows: total volume of the PCR reaction 50 μL, wherein template DNA 2 μL, upstream primer and downstream primer (10 uM) 1 μL, respectively, dNTP (2.5 mM for each four dNTP) 4 μL, 5× Prime Star buffer (Mg2+ Plus) 10 μL, Prime Star (5 U/ul) 0.5 μL, and dilute with ddH2O to the total volume of 50 μL. Cycle parameters: predenaturation at 94° C. 3 min, then 94° C. 30 s, 57° C. 15 s, 72° C. 2 min for 45 cycles, extending at 72° C. for 10 min. The product is recycled and purified via agarose gel electrophoresis with the concentration of 1%.
Meanwhile, take the expression plasmid HBV1.37 which contained HBV 1.3-fold complete sequence in laboratory as a template, to amplify the above three pieces of HBV gene segment. The product is recycled and purified via agarose gel electrophoresis with the concentration of 1%, for the quality control of the high-throughput sequencing.
The purified PCR product performs sanger sequencing, and three fragments is pieced for a whole HBV DNA complete sequence by DNASTAR software.
Sequencing method: the purified PCR product is sequenced by nano institute, Zhejiang university, the experiment process is briefly described as follows: 1, the genomic DNA is disrupted into small fragments of 200 bp, both ends of each fragment add connectors (each sample add a specific connector). 2, DNA fragments are purified by QIAGEN gel extraction kit. 3, PCR amplifies the DNA fragment with connectors and is purified. 4, detected by Solexa high-throughput sequencing platform.
Data Analysis (1) Splicing of Sequencing Fragment (Mapping)
Obtain the sequence (reads, tags) data of each sample by the high-throughput sequencing, firstly the sequence data in both directions is composed into a file, and then is spliced into a complete HBV genome by using bowtie software. The Chinese HBV genome standard type B and C sequences reported in “Profile of public nucleotide databases HBV complete genome sequence and establishment of Chinese HBV reference sequence” (Progress in Natural Science 2008, 18 (2)) acts as splicing reference sequence (SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11), while a length of sequence is extracted before and after the gap of the linear genome to compose into a short fragment and the reference sequence is added as well, to ensure the sequence covering on the gap can be correctly spliced. Parameter is set as: seed length=30, allowable mismatch=3. Since according to the principle of high-throughput sequencing, the results of the previous test has the highest accuracy, with the sequencing length increases, the accuracy decreases, thus we choose the first 30 bases for comparison, the sequence with more than three mutations is considered as a sequence with relatively poor sequencing quality, and is removed (i.e., every 10 bases allow a mutation)
(2) Statistical Tests of the Composition Difference of Bases at Each Locus
Perform statistics on the sequence of the splicing file of each sample and its coordinate read in the genome, to obtain the absolute number of the four types of bases measured in all base positions of the complete genome. Then each sample of the severe hepatitis B group and mild hepatitis B group are compared in turn by using combination method. Obtain the base type with significant difference in each locus between two samples (p<0.001) by applying Chi-square test; perform statistics on the above information in all comparison pairs, and take the base type detected with significant difference in more than a half of comparison pairs, while without opposite significant proportion change of base at a certain locus in any comparison pair as the base change with significant difference between the severe hepatitis B group and mild hepatitis B group, and remains (the above steps are completed by writing a script by Python program language (www.python.org)).
Through the statistical analysis of the sequencing results of the severe hepatitis B and mild hepatitis B, two new mutation loci are found that: 216T→C and 285G→A. For the 12 cases of severe hepatitis patients, 10 cases exist 216T→C mutation, while 9 cases exist 285G→A mutation.
Though Solexa high-throughput sequencing, it has been detected that, in the severe hepatitis patients with 216T→C mutation, the proportion of 216T→C mutant strain is 8% to 100%; while in the severe hepatitis patients with 285G→A mutation, the proportion of 285G→A mutant strain is 11% to 100%. In the 12 cases of severe hepatitis patients, only 1 case exists duplex mutation of 216T→C and 285G→A, and the proportion of 216T→C mutant strain and 285G→A mutant strain is 17% and 7%, respectively. Further, it is found that in this experiment, when the proportion of 216T→C mutant strain is less than 17%, and the proportion of 285G→A mutant strain is less than 11%, those mutant strains are unable to be detected by conventional sanger sequencing method, such that this type of case is unable to be detected as well.
The results showed that, the solexa high-throughput sequencing technology features much better sensitivity of mutation detection than that of the conventional sanger sequencing, and enables to find the non-dominant strains existed in HBV patients, and is beneficial for us to study the complex quasispecies characteristics in patients. While sanger sequencing method is reliable, accurate, and forms large scale, and is still a best choice for a small number of sequences, thereby commonly being used for the sequencing of PCR products, terminal sequencing of the plasmids and bacterial artificial chromosomes.
In addition, the majority of patients with severe hepatitis have 216T→C and 285G→A mutations, the mutations of the two loci results in a change of amino acid of HBV S region, as shown in the table below:
Therefore, 216T→C and 285G→A mutations of hepatitis B virus may be related with the onset of severe hepatitis, further research would offer help for clinical early intervention and diagnostic.
First: Serum Collection in Patients with Hepatitis B
Collect serum samples of hepatitis B cases from a plurality of domestic hospitals, wherein there are 102 cases with severe hepatitis B, 127 cases with chronic mild hepatitis B, 95 cases with chronic moderate and severe hepatitis B and 65 cases of carriers (the patients has been diagnosed explicitly with hepatitis B by the indicators of ALT, AST, TBIL, PT, viral titer, etc., and clinical manifestations))
We download 616 complete sequences of HBV genome, and design a pair of primer (SEQ ID NO: 8, SEQ ID NO: 9) by using primer 5.0 software according to the complete genome conserved sequence of HBV, to amplify a length of gene fragment including No. 216 and No. 285 of the HBV genome sequence.
Assemble the above primer (SEQ ID NO: 8, SEQ ID NO: 9) and dNTP, 5× Prime Star buffer, Prime Star, ddH2O for a HBV mutant amplification kit.
An equal volume of nucleic acid extract is added into the 50u1 serum and mixed, followed by water bath at 100 for 10 min, centrifugation for 10 min with 13000 r/min, and then the supernatant is drawn for PCR amplification.
The brief steps of the amplification for the HBV gene of a patient are as follows: total volume of the PCR reaction 50 μL, wherein template DNA 2 μL, primers (10 uM) 1 μL, respectively, dNTP (2.5 mM for each) 4 μL, 5×Prime Star buffer (Mg2+ Plus) 10 μL, Prime Star (5 U/ul) 0.5 μL, and dilute with ddH2O to the total volume of 50 μL. Cycle parameters: predenaturation at 94° C. 3 min, then 94° C. 30 s, 56° C. 15 s, 72° C. 50 s for 45 cycles, extending at 72° C. for 10 min. The product is recycled and purified via agarose gel electrophoresis with the concentration of 1%.
The purified PCR product performs sequencing according to the conventional sanger sequencing technique in the art.
Seventh: Statistical Method Apply Nonparametric Rank Sum Test, p<0.05 is Considered as a Statistically Significant Difference.
Specific experimental results are shown in Table 1-3. The data from table 1 and table 2 show that: the incidence of 216T→C mutation in patients with severe hepatitis is 45% (46/102), while the incidence of 285G→A mutation is 39% (40/102); loci mutation frequency of 216T→C in patients with chronic moderate and severe hepatitis is 23% (22/95), while the incidence of 285G→A mutation is 29% (28/95); loci mutation frequency of 216T→C in patients with chronic mild hepatitis is 8% (10/127), while the incidence of 285G→A mutation is 5% (6/127); loci mutation frequency of 216T→C in hepatitis B carriers is 5% (3/65), while 285G→A mutation is not found. Compare the hepatitis disease between the mutational sample and non-mutational sample by applying nonparametric rank sum test, showing the result of P<0. 001, which indicates that mutational case tends to occur severe inflammation of the liver.
Table 3 shows that: the incidence of 216T→C/285G→A dual mutation in patients with severe hepatitis is 31% (32/102), the incidence in patients with chronic moderate and severe hepatitis is 16% (15/95), the incidence in patients with chronic mild hepatitis is 4% (5/127), and the incidence in carriers is 0%; nonparametric rank sum test shows P<0. 001, the experimental data indicate that 216T→C/285G→A duplex mutational case tends to have severe inflammation of the liver.
The experimental results show that: the 216T→C and 285G→A mutations are closely related to the aggravated liver inflammation, and their further study is beneficial for clarifying the molecular mechanism of hepatitis B aggravation and has positive significance for clinical early warning of severe hepatitis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201110384731.7 | Nov 2011 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2012/071108 | 2/14/2012 | WO | 00 | 8/18/2014 |
