A primer pair and its application in the quality control method of BCG-CpG-DNA adjuvant

Abstract

The present invention relates to a primer pair and its application in the quality control method of BCG-CpG-DNA adjuvant. The present invention first uses the BCG genomic DNA fragment itself as a quality control fragment, verifies its feasibility, amplifies the quality control fragment with specific primers, analyzes the amplification results, and thus conducts quality control on the BCG-CpG-DNA adjuvant.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, especially the quality control of vaccine adjuvants. Specifically, the present invention relates to a primer pair and its application in the quality control method of BCG-CpG-DNA adjuvant. Background Art

[0002] Adjuvants play an essential role in vaccines. In terms of the immune function of adjuvants, the earliest used aluminum adjuvant, as the only widely used vaccine adjuvant at present, its safety is beyond doubt, but it can only enhance humoral immunity and cannot enhance cellular immunity. Entering the new era, due to the development of new technologies such as molecular purification, recombinant technology, and analytical biochemistry, and the in-depth understanding of the immune mechanism, there is an increasing urgency to develop new immune adjuvants.

[0003] In recent years, researchers have conducted a large number of studies to find more efficient and safe new adjuvants. Many experiments have confirmed that bacterial DNA has an immune-enhancing effect on cellular immunity in vitro. Currently, more studied CpG-DNA is DNA containing non-methylated CpG (cytosine-guanine dinucleotide) motifs, which has strong immune-enhancing activity. The research results using synthetic CpG oligonucleotides as adjuvants show that they have the dual effects of enhancing cellular immunity and humoral immunity. BCG-CpG-DNA is natural CpG-DNA extracted from Bacillus Calmette-Guérin (BCG), which has good immune-stimulating activity of CpG, and BCG has been used worldwide for many years, proving that this adjuvant has good safety. CpG-DNA adjuvant has a synergistic effect with aluminum adjuvant and is a promising new adjuvant that can be used as an adjuvant in preventive and therapeutic vaccines.

[0004] However, BCG-CpG-DNA adjuvant is a nucleic acid fragment with a molecular weight distribution of 2-10 kb obtained by mechanically and randomly breaking natural BCG cells, followed by ion exchange and displacement chromatography, and finally ultrafiltration through a hollow fiber column. The existing quality control indicators mainly measure the content of non-methylated CpG, nucleic acid content, and purity, which cannot fully reflect the distribution of different molecular weights of CpG-DNA adjuvant, that is, the quality control research on the distribution range of 2-10 kb molecular weight fragments has not been carried out, and DNA fingerprinting and quantitative quality control need to be carried out.

[0005] Disadvantages of the existing technology: (1) BCG-CpG-DNA adjuvant is a DNA fragment extracted from broken BCG cells, and the mechanical fragmentation used has randomness, making it difficult to establish and control the batch-to-batch differences; (2) Currently, the quality control indicators of this adjuvant only include CpG content and nucleic acid purity. In the existing methods, methylation treatment of samples and complex operations such as high-performance liquid chromatography are required. This quality control method has the disadvantages of cumbersome operation steps, extremely high requirements for the experimental techniques of operators, and long time-consuming cycles.

[0006] Currently, no effective quality control of BCG-CpG-DNA adjuvant using long-chain PCR amplification technology has been found in the publicly available data, and the uniformity of its nucleic acid distribution has not been evaluated. Summary of the Invention

[0007] To overcome the above defects, the present invention provides a primer pair and its application in the quality control method of BCG-CpG-DNA adjuvant. Specifically,

[0008] In the first aspect of the present invention, a primer pair is provided, wherein the primers in the primer pair have at least 29 bp and are used to amplify a quality control fragment in the BCG-CpG-DNA adjuvant. The quality control fragment is determined according to the genomic DNA of Mycobacterium bovis BCG, and the quality control fragment is a genomic DNA fragment of Mycobacterium bovis BCG with a size of 2 - 6.0 kb.

[0009] Preferably, the size of the quality control fragment includes 2 - 6.0 kb, such as 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, 5.0 kb, 5.5 kb, 6.0 kb, or any range of the above values, such as 3.0 - 6.0 kb, 3.0 - 5.0 kb, etc.

[0010] Preferably, the primer has a high Tm value, and the Tm value is at least 60 °C.

[0011] Preferably, the primer pair includes one or more pairs and is used to amplify one or more quality control fragments.

[0012] More preferably, the multiple quality control fragments include, for example, at least 2 fragments, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.; more preferably, the quality control fragments include 2 - 10, further preferably 3 - 7, such as 5 quality control fragments.

[0013] More preferably, when the primer pair is multiple pairs, the quality control fragments that can be amplified have the same or different sizes.

[0014] More preferably, the quality control fragments have the same size and are divided into 4 - 7 segments according to the genomic DNA of Mycobacterium bovis BCG, preferably 5 segments, and the quality control fragments of 3.0 kb, 5.0 kb, and / or 6.0 kb are amplified in each segment.

[0015] Alternatively, the quality control fragments have different sizes, and quality control fragments with different sizes can be selected within the same segment or different segments.

[0016] Further preferably, the same upstream primer is used to amplify the quality control fragments with different sizes in each segment.

[0017] (1) SEQ ID No:1 and SEQ ID No:2;

[0018] (2) SEQ ID No:1 and SEQ ID No:3;

[0019] (3) SEQ ID No:1 and SEQ ID No:4;

[0020] (4) SEQ ID No:6 and SEQ ID No:7;

[0021] (5) SEQ ID No:6 and SEQ ID No:8;

[0022] (6) SEQ ID No:6 and SEQ ID No:9;

[0023] (7) SEQ ID No:11 and SEQ ID No:12;

[0024] (8) SEQ ID No:11 and SEQ ID No:13;

[0025] (9) SEQ ID No:11 and SEQ ID No:14;

[0026] (10) SEQ ID No:16 and SEQ ID No:17;

[0027] (11) SEQ ID No:16 and SEQ ID No:18;

[0028] (12) SEQ ID No:16 and SEQ ID No:19;

[0029] (13) SEQ ID No:21 and SEQ ID No:22;

[0030] (14) SEQ ID No:21 and SEQ ID No:23; and / or,

[0031] (15) SEQ ID No:21 and SEQ ID No:24.

[0032] Preferably, the primer pair includes any one or more combinations of the above.

[0033] For example, the primer pair can be used to amplify a quality control fragment of 3.0 kb, 5.0 kb or 6.0 kb in any region. Or,

[0034] The primer pair can be used to amplify all 3.0 kb quality control fragments in 5 segments, all 5.0 kb quality control fragments in 5 segments, or all 6.0 kb quality control fragments in 5 segments. Or,

[0035] any combination of the above quality control fragments.

[0036] In a second aspect of the present invention, there is provided an application of a quality control fragment or its detection reagent in a BCG-CpG-DNA adjuvant for quality control of the BCG-CpG-DNA adjuvant, wherein the quality control fragment is determined according to the genomic DNA of Mycobacterium bovis BCG, and the quality control fragment is a genomic DNA fragment of Mycobacterium bovis BCG with a size of 2 - 6.0 kb.

[0037] Preferably, the size of the quality control fragment includes 2 - 6.0 kb, such as 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, 5.0 kb, 5.5 kb, 6.0 kb, or any range of the above values, such as 3.0 - 6.0 kb, 3.0 - 5.0 kb, etc.

[0038] Preferably, the quality control fragment includes one or more, such as at least 2 fragments, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.; more preferably, the quality control fragment includes 1 - 10, and further preferably 1 - 5 quality control fragments.

[0039] More preferably, the quality control fragments have the same or different sizes.

[0040] More preferably, the quality control fragments are the same, and according to the genomic DNA of Mycobacterium bovis BCG, they are divided into 4 - 7 segments, preferably 5 segments, and 3.0 kb, 5.0 kb and / or 6.0 kb quality control fragments of each segment are amplified.

[0041] Further preferably, the quality control fragments are from the same or different segments.

[0042] Preferably, the detection reagent includes a primer pair for amplifying the quality control fragment.

[0043] More preferably, the primer pair is defined as in the first aspect of the present invention.

[0044] In a third aspect of the present invention, there is provided a quality control method for a BCG-CpG-DNA adjuvant, the quality control method comprising,

[0045] 1) determining a quality control fragment according to the genomic DNA of Mycobacterium bovis BCG, the quality control fragment being a genomic DNA fragment of Mycobacterium bovis BCG with a size of 2 - 10.0 kb;

[0046] 2) Detect the quality control fragments;

[0047] 3) Analyze the detection results of the quality control fragments in different batches of BCG-CpG-DNA adjuvant.

[0048] Preferably, the size of the quality control fragments in step 1) includes 2 - 10.0 kb, such as 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb, 4.5 kb, 5.0 kb, 5.5 kb, 6.0 kb, or any range of the above values, such as 3.0 - 6.0 kb, 3.0 - 5.0 kb, etc.

[0049] Preferably, the quality control fragments in step 1) include one or more quality control fragments, such as at least 2 fragments, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.; more preferably, the multiple quality control fragments in step 2 include 1 - 10, and further preferably 1 - 5 quality control fragments.

[0050] Preferably, when the quality control fragments in the step are multiple, the sizes of the quality control fragments are the same or different.

[0051] In a specific embodiment, the size of the quality control fragments is 3.0 kb, 5.0 kb, and / or 6.0 kb.

[0052] Preferably, when the quality control fragments are multiple, the nucleotide sequences of the quality control fragments are the same or different.

[0053] It can be divided into different segments according to the BCG genomic DNA sequence, and different quality control fragments are selected for different segments. For example, the BCG genomic DNA sequence is divided into 4 - 7 segments, and different 3.0 kb, 5.0 kb, and / or 6.0 kb fragments are selected as quality control fragments for each segment.

[0054] Alternatively, since the sizes of the quality control fragments are different, quality control fragments with different sizes can be selected within the same segment, or segments with different sizes can be selected in different segments.

[0055] In a specific embodiment, the BCG genomic DNA sequence is divided into 5 segments, and one or more quality control fragments among 3.0 kb, 5.0 kb, and / or 6.0 kb in each segment are detected.

[0056] More preferably, the quality control fragments with different sizes in the same segment have upstream sequences with the same structure.

[0057] Preferably, the detection method in step 2) includes long-chain PCR technology and fluorescence quantitative PCR to detect the quality control fragments.

[0058] More preferably, the long-chain PCR method includes designing multiple pairs of primer pairs for a plurality of quality control fragments and amplifying the above different quality control fragments.

[0059] Even more preferably, the primer pairs are defined as in the first aspect of the present invention.

[0060] Preferably, the analysis of the detection results in step 3) includes evaluating the homogeneity of the quality control fragments of different batches of BCG-CpG-DNA by gray scale scanning and relative quantification ratio.

[0061] Even more preferably, the analysis of the detection results includes expressing the relative expression levels of the quality control fragments of other batches relative to the reference band with the expression level of the quality control fragments of a certain batch as the reference band.

[0062] Advantageous technical effects of the present invention:

[0063] 1. For the first time, the feasibility of using the BCG genomic DNA fragment itself as a quality control fragment is verified. There is no need for complex operations such as methylation treatment of adjuvants and high performance liquid chromatography. The method of the present invention is simple and feasible. The quality control method of the present invention improves the efficiency of quality control and reduces the cost;

[0064] 2. The present invention adopts long-chain polymerase chain reaction (PCR) technology. Based on the fact that the whole genome sequencing of the strain for the production of BCG-CpG-DNA adjuvant has been obtained, multiple pairs of specific primers are designed by sequentially selecting gene sequences at five regions from the 5'→3' end of the whole genome. Through long-chain PCR technology, different quality control fragments (3 kb, 5 kb and / or 6 kb) are amplified, improving the specificity of detection. The method can be used for the quality control of the uniform nucleic acid distribution of BCG-CpG-DNA adjuvant.

[0065] The above only summarizes some aspects of the present invention and should not be considered as limiting the present invention in any aspect.

[0066] All patents and publications mentioned in this specification are incorporated into the present invention by reference as a whole. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the concept or scope of the present invention.

[0067] The following examples further illustrate the present invention in detail and should not be considered as limiting the present invention or the scope of the specific methods described by the present invention. Brief Description of the Drawings

[0068] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0069] Figure 1The agarose gel electrophoresis results of the PCR amplification products of the 3 kb quality control fragments of different segments using 7 batches of BCG-CpG-DNA adjuvant as templates are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively.

[0070] Figure 2 The agarose gel electrophoresis results of the PCR amplification products of the 5 kb quality control fragments of different segments using 7 batches of BCG-CpG-DNA adjuvant as templates are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively.

[0071] Figure 3 The agarose gel electrophoresis results of the PCR amplification products of the 6 kb quality control fragments of different segments using 7 batches of BCG-CpG-DNA adjuvant as templates are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively.

[0072] Figure 4 The agarose gel electrophoresis results of the PCR amplification products of the 7 kb quality control fragments of different segments using 7 batches of BCG-CpG-DNA adjuvant as templates are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively.

[0073] Figure 5 The gray scale scanning results of the PCR products of the 3 kb quality control fragment primers of different segments are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively.

[0074] Figure 6 The gray scale scanning results of the PCR products of the 5 kb quality control fragment primers of different segments are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively.

[0075] Figure 7 The gray scale scanning results of the PCR products of the 6 kb quality control fragment primers of different segments are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively.

[0076] Figure 8 The gray scale scanning results of the PCR products of the 7 kb quality control fragment primers of different segments are shown, where (a) to (e) represent the 1 / 5 to 5 / 5 segments respectively. Detailed implementation manners

[0077] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and such modifications and substitutions all fall within the protection scope of the present invention.

[0078] In each of the following examples, the equipment and materials were obtained from the several companies indicated below:

[0079] Dual 48-well rapid PCR instrument (Bio-Rad, USA);

[0080] Nucleic acid horizontal electrophoresis tank (Beijing Liuyi Instrument Co., Ltd.);

[0081] Fully automatic stain-free gel imaging system (Bio-Rad, USA);

[0082] PCR amplification reagents such as LA Taq and DNA Marker (Takara Bio Inc. (Beijing));

[0083] Primers (Sangon Biotech (Shanghai) Co., Ltd.).

[0084] Example 1

[0085] In the quality control method of the BCG-CpG-DNA adjuvant of the present invention, according to the completed genomic DNA sequence information of Mycobacterium bovis BCG, primer pairs were designed using the prime 5.0 software, and then a suitable thermal cycling reaction system was screened out. Using the long-chain PCR amplification technique, PCR amplification was carried out under certain reaction conditions. The PCR products were detected by agarose gel electrophoresis to determine the size of the target quality control molecular weight. The electrophoresis pattern was scanned by Image Lab 3.0, and the relative gray-scale ratio of the other lane segments to the reference band was compared, and the gray-scale ratio of the quality control fragments of the same molecular weight in each segment was analyzed and compared.

[0086] The specific implementation plan is as follows:

[0087] 1. Primer pair design: According to the genomic sequence of Mycobacterium bovis BCG, it was evenly divided into five segments from the 5' end to the 3' end, and then primer pairs were designed according to the sequences of each segment. The sizes of the target quality control fragments amplified in each segment were approximately 3 kb, 5 kb, 6 kb, and 7 kb respectively. The specific primers are as follows:

[0088] Table 1: Primer sequence information

[0089]

[0090]

[0091]

[0092] 2. Preparation of PCR amplification template: The BCG-CpG-DNA adjuvant was 1 mg / ml, and the adjuvant was diluted 100-fold to obtain the PCR amplification template. When used, 5 μl was added as the DNA template according to a 50 μl reaction volume.

[0093] 3. Obtaining quality control fragments of each molecular weight by PCR amplification: Using the diluted BCG-CpG-DNA adjuvant above as a template, the following reaction system was composed: 1×LA PCR buffer (Mg 2+ Free), 2.5 mM MgCl2, 0.4 mM dNTP, 2.5 U of LA Taq, 20 μM each of the upstream and downstream primers, 50 ng of DNA template, and the balance was water. The negative control was water without DNA template.

[0094] 4. The PCR amplification program was: pre-denaturation at 94 °C for 1 min, 10 cycles (98 °C for 20 s, 68 °C for 4 min), 15 cycles (98 °C for 20 s, 68 °C (4 - 8) min + 15 s / cycle), 72 °C for 10 min; incubation at 4 °C.

[0095] 5. Detecting the amplified products by agarose gel electrophoresis and gray-scale scanning

[0096] Take 3 μl of the product and perform electrophoresis on a 1.0% agarose gel, take a photo and save it. Perform gray-scale scanning on the agarose gel electrophoresis using Image Lab 3.0.

[0097] Specifically:

[0098] Using the BCG-CpG-DNA adjuvant of 7 batches as a template and the upstream and downstream primers of the 3 kb quality control molecular weight fragment in the 1 / 5 - 5 / 5 section, perform long-chain PCR. The amplification program is as described in 4. PCR amplification program. After PCR, take 5 μl of the PCR product, add 1 μl of 6×Loading Buffer, mix evenly and load the sample. Detect by agarose gel electrophoresis, and the results are as Figure 1 (a)- Figure 1 (e):

[0099] The agarose gel electrophoresis detection results of other quality control fragments of 5, 6, and 7 kb in the 1 / 5 - 5 / 5 section are shown in Figures 2 - 4 .

[0100] Taking the 3 kb quality control fragment in the 1 / 5 section as an example, the PCR amplification map was subjected to gray-scale scanning using Image Lab 3.0 software. Lane 4 was used as the reference band (recorded as a gray value of 1.00). Lanes 1 - 3 and 5 - 7 were the agarose gel electrophoresis results of the PCR amplification products using different batches of adjuvants as templates. The corresponding gray values were 0.90, 1.11, 1.03, 0.99, 1.16, and 0.95 (see Figure 5 (a)).

[0101] Gray-scale scanning results: Using one batch as the reference band, the gray-scale ratio of the PCR products of the primer pairs in the 1 / 5 section of the 3 kb quality control fragment with other batches of CpG adjuvant as the template to the reference band in the agarose gel electrophoresis is between 0.90 and 1.16, that is, it is basically consistent with the gray-scale ratio of the reference band. It can realize the quality control of the homogeneity detection of different fragments of BCG-CpG-DNA adjuvant nucleic acid.

[0102] The relative gray-scale scanning results of the quality control fragments in other quality control fragments are specifically as Figures 5 - 8 and Table 2:

[0103] Table 2: Gray-scale scanning results of different quality control fragments in different sections

[0104]

[0105]

[0106] The above results show that the fragments of BCG genomic DNA, especially 3.0 kb, 5.0 kb, and 6.0 kb, are relatively homogeneous in the adjuvant, and the difference between the highest and lowest relative values does not exceed 1.0, which can well evaluate the stability of BCG-CpG-DNA adjuvant or the homogeneity of nucleic acid, making it feasible to use it as a quality control fragment. However, since the 7 kb DNA fragment is too large, it is not uniformly present in the BCG-CpG-DNA adjuvant after mechanical fragmentation, and the difference between different batches is relatively large, with the difference between the highest and lowest values being greater than 1.0, so it is not suitable as a quality control index.

[0107] The present invention evaluates the homogeneity of the distribution of different batches of BCG-CpG-DNA adjuvant through different quality control molecular weight fragments. The expected result is that using different batches of adjuvant as the template, specific target bands can be amplified for different quality control fragments in each section, and using one batch as the reference band, the relative quantitative ratio of the gray-scale scanning of the target bands of other batches is within a certain range. The results suggest that it is feasible to use the BCG genomic DNA fragment itself as a quality control fragment. The nucleic acid molecular weight distribution of different batches of adjuvant is in good homogeneity.

[0108] At the same time, the method of the present invention is convenient to operate, the data is visual, and the evaluation is convenient.

[0109] This implementation scheme is only a conventional PCR amplification. The entire test cycle takes about 5 h, while the previous methylation operation, from sample processing to high-performance liquid chromatography separation operation, takes about 24 h. The method of the present invention improves the quality control efficiency and reduces the cost.

[0110] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0111] In addition, it should be noted that, in the various specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0112] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. Application of primer pair in quality control of BCG-CpG-DNA adjuvant, characterized in that, The primer pairs include: (1) SEQ ID No: 1 and SEQ ID No: 2; (2) SEQ ID No: 1 and SEQ ID No: 3; (3) SEQ ID No: 1 and SEQ ID No: 4; (4) SEQ ID No: 6 and SEQ ID No: 7; (5) SEQ ID No: 6 and SEQ ID No: 8; (6) SEQ ID No: 6 and SEQ ID No: 9; (7) SEQ ID No: 11 and SEQ ID No: 12; (8) SEQ ID No: 11 and SEQ ID No: 13; (9) SEQ ID No: 11 and SEQ ID No: 14; (10) SEQ ID No: 16 and SEQ ID No: 17; (11) SEQ ID No: 16 and SEQ ID No: 18; (12) SEQ ID No: 16 and SEQ ID No: 19; (13) SEQ ID No: 21 and SEQ ID No: 22; (14) SEQ ID No: 21 and SEQ ID No: 23; and (15) SEQ ID No: 21 and SEQ ID No:

24.

2. A quality control method for BCG-CpG-DNA adjuvant, characterized in that, The quality control method includes 1) Using the primer pairs described in claim 1 and using the long-chain PCR method to detect the BCG-CpG-DNA adjuvant; 2) Analyzing the detection results of different batches of BCG-CpG-DNA adjuvant; The analysis of the detection results includes evaluating the homogeneity of different batches of BCG-CpG-DNA adjuvant by gray-scale scanning and relative quantitative ratio.

Images