阅读说明：本技术 一种扩增鼠单抗重轻链基因序列的方法及其引物和筛选该引物的方法 (Method for amplifying heavy-light chain gene sequence of mouse monoclonal antibody, primer thereof and method for screening primer ) 是由 严行波 陈玲 尉宁 于 2020-12-16 设计创作，主要内容包括：本申请公开了一种扩增鼠单抗重轻链基因序列的方法及其引物,以及筛选所述引物的方法,其中,各复合引物所用的正向引物均为组合物,所述复合引物通过预先筛除可扩增非功能性基因序列的引物对而得,并且,每组正向引物均已剔除扩增Sp2/0细胞非功能轻链基因的引物,采用本申请提供的复合引物进行PCR扩增,所得引物无需做TA克隆,而是能够满足测序的纯度要求,进一步地,采用本申请提供的复合引物能够减少扩增管的数量,简化扩增操作,使PCR扩增的操作更为简单方便。(The application discloses a method for amplifying a murine monoclonal antibody heavy and light chain gene sequence, primers thereof and a method for screening the primers, wherein forward primers used by all composite primers are all compositions, the composite primers are obtained by screening out primer pairs capable of amplifying non-functional gene sequences in advance, each group of forward primers are provided with a primer for rejecting and amplifying Sp2/0 cell non-functional light chain genes, the composite primers provided by the application are used for carrying out PCR amplification, the obtained primers do not need to be subjected to TA cloning but can meet the purity requirement of sequencing, further, the composite primers provided by the application can reduce the number of amplification tubes, simplify amplification operation and enable the operation of PCR amplification to be simpler and more convenient.)

1. A first composite primer for amplification of a mouse monoclonal antibody kappa chain, wherein the first composite primer for amplification of a mouse monoclonal antibody kappa chain comprises a first forward amplification primer set KFA and a kappa chain reverse amplification primer KR, wherein the KFA comprises the following primers:

KFA 1: the nucleotide sequence is shown as SEQ ID NO. 1;

KFA 2: the nucleotide sequence is shown as SEQ ID NO. 2;

KFA 3: the nucleotide sequence is shown as SEQ ID NO. 3;

KFA 4: the nucleotide sequence is shown as SEQ ID NO. 4;

KFA 5: the nucleotide sequence is shown as SEQ ID NO. 5;

KFA 6: the nucleotide sequence is shown as SEQ ID NO. 6; and

KFA 7: the nucleotide sequence is shown as SEQ ID NO. 7;

the nucleotide sequence of KR is shown in SEQ ID NO. 8.

2. A second composite mouse monoclonal antibody kappa chain amplification primer, wherein the second composite mouse monoclonal antibody kappa chain amplification primer comprises a kappa chain second forward amplification primer set KFB and a kappa chain reverse amplification primer KR, and the KFB comprises the following primers:

KFB1, the nucleotide sequence of which is shown in SEQ ID NO. 9;

KFB2, the nucleotide sequence of which is shown in SEQ ID NO. 10;

KFB3, the nucleotide sequence of which is shown in SEQ ID NO. 11;

KFB4, the nucleotide sequence of which is shown in SEQ ID NO. 12;

KFB5, the nucleotide sequence of which is shown in SEQ ID NO. 13;

KFB6, the nucleotide sequence of which is shown in SEQ ID NO. 14;

KFB7, the nucleotide sequence of which is shown in SEQ ID NO. 15;

KFB8, the nucleotide sequence of which is shown in SEQ ID NO. 16;

KFB9, the nucleotide sequence of which is shown in SEQ ID NO. 17;

KFB10, the nucleotide sequence of which is shown in SEQ ID NO. 18;

KFB11, the nucleotide sequence of which is shown in SEQ ID NO. 19;

KFB12, the nucleotide sequence of which is shown in SEQ ID NO. 20;

KFB13, the nucleotide sequence of which is shown in SEQ ID NO. 21; and

KFB14, the nucleotide sequence of which is shown in SEQ ID NO. 22;

the nucleotide sequence of KR is shown in SEQ ID NO. 8.

3. The composite amplification primer for the lambda chain of the murine monoclonal antibody is characterized by comprising a lambda chain forward amplification primer group LFC and a lambda chain reverse amplification primer LR, wherein the LFC comprises:

LFC1, the nucleotide sequence of which is shown in SEQ ID NO. 23; and

LFC2, the nucleotide sequence of which is shown in SEQ ID NO. 24;

the LR has a nucleotide sequence shown in SEQ ID NO. 25.

4. A mouse monoclonal antibody heavy chain amplification composite primer is characterized by comprising a heavy chain forward amplification primer group HFA and a heavy chain reverse amplification primer HR, wherein the HFA comprises:

HFA1, the nucleotide sequence of which is shown in SEQ ID NO. 26;

HFA2, the nucleotide sequence of which is shown in SEQ ID NO. 27;

HFA3, the nucleotide sequence of which is shown in SEQ ID NO. 28;

HFA4, the nucleotide sequence of which is shown in SEQ ID NO. 29;

HFA5, the nucleotide sequence of which is shown in SEQ ID NO. 30;

HFA6, the nucleotide sequence of which is shown in SEQ ID NO. 31;

HFA7, the nucleotide sequence of which is shown in SEQ ID NO. 32;

HFA8, the nucleotide sequence of which is shown in SEQ ID NO. 33;

HFA9, the nucleotide sequence of which is shown in SEQ ID NO. 34;

HFA10, the nucleotide sequence of which is shown in SEQ ID NO. 35;

HFA11, the nucleotide sequence of which is shown in SEQ ID NO. 36;

HFA12, the nucleotide sequence of which is shown in SEQ ID NO. 37;

HFA13, the nucleotide sequence of which is shown in SEQ ID NO. 38;

HFA14, the nucleotide sequence of which is shown in SEQ ID NO. 39; and

HFA15, the nucleotide sequence of which is shown in SEQ ID NO. 40;

the HR is derived from the common gene sequence of mouse IgG1 antibody, IgG2a antibody, IgG2b antibody and IgG3 antibody in the constant region CH 1.

5. An amplification kit comprising at least one of the primers of claims 1 to 4.

6. A method of obtaining a murine single heavy light chain, comprising:

and (2) respectively amplifying a light chain gene and a heavy chain gene by using a murine cDNA as a template and using a murine monoclonal antibody light chain primer and a murine monoclonal antibody heavy chain primer, wherein the murine monoclonal antibody light chain primer is as defined in any one of claims 1 to 3, and the murine monoclonal antibody heavy chain primer is as defined in claim 4.

7. The method of claim 6, wherein the murine mab is an IgG type murine mab.

8. The method of claim 6 or 7, further comprising, after amplifying the light chain genes and the heavy chain genes:

performing antibody sequencing by using the light chain gene sample and the heavy chain gene sample obtained by amplification;

and searching the genome position of the detected antibody sequence by using a database, and determining the leader peptide sequence of the detected antibody sequence.

9. A method of screening for the composite primer, the method comprising:

performing PCR amplification by using a plurality of pairs of candidate primer pairs respectively by using sp2/0cDNA as a template;

and (3) carrying out electrophoretic detection on the PCR amplification result of each pair of candidate primer pairs, and rejecting the candidate primer pairs capable of amplifying the sp2/0cDNA non-functional gene sequence.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for amplifying gene sequences of heavy and light chains of a monoclonal antibody in a mouse hybridoma cell, primers of the method, and a method for screening the primers.

Background

The monoclonal antibody has wide application In the biomedical field and plays an important role In the fields of In Vitro Diagnosis (IVD) and the like. Obtaining antibody heavy and light chain sequences from hybridoma cells through a gene sequencing technology is the first step of performing operations such as monoclonal antibody resource permanent storage, constructing recombinant antibodies, performing antibody in-vitro affinity maturation and the like.

Sp2/0 cells used for hybridoma cell fusion contain endogenous non-functional genes that are not efficiently translated into protein, and therefore, when antibody light chain gene sequences are amplified from hybridoma cells, the endogenous non-functional gene sequences produce false positive interference.

Currently, various strategies are used to amplify light chain antibody genes from hybridoma cells, the most common of which is the removal of endogenous non-functional genes by restriction endonuclease methods, but such methods are complicated to operate and are inefficient; another method is to construct TA clone from the amplified product, and then sequence and identify a plurality of clones obtained by purifying the TA clone respectively to eliminate endogenous non-functional genes, but the method is time-consuming and labor-consuming.

Disclosure of Invention

The method uses the specific composite primer, can avoid false positive interference generated by endogenous non-functional genes, obtains purer products by amplification by the method, has higher content of target amplification products, and can be directly used for sequencing research.

The application aims to provide a mouse monoclonal Antibody kappa chain amplification first composite primer, which is used for amplifying a mouse monoclonal Antibody kappa chain, and particularly comprises a first forward amplification primer group KFA and a kappa chain reverse amplification primer KR, wherein the KFA is derived from Antibody engineering.

In one realizable approach, the KFA includes the following primers:

KFA1, the nucleotide sequence of which is shown in SEQ ID NO. 1;

KFA 2: the nucleotide sequence is shown as SEQ ID NO. 2;

KFA 3: the nucleotide sequence is shown as SEQ ID NO. 3;

KFA 4: the nucleotide sequence is shown as SEQ ID NO. 4;

KFA 5: the nucleotide sequence is shown as SEQ ID NO. 5;

KFA 6: the nucleotide sequence is shown as SEQ ID NO. 6; and

KFA 7: the nucleotide sequence is shown in SEQ ID NO. 7.

Further, the primers in KFA were combined in equal amounts.

Further, the nucleotide sequence of KR is shown in SEQ ID NO. 8.

It is another object of the present application to provide a mouse monoclonal antibody kappa chain amplification second composite PRIMER that is also used for amplifying a mouse monoclonal antibody kappa chain, specifically, the mouse monoclonal antibody kappa chain amplification second composite PRIMER includes a kappa chain second forward amplification PRIMER set KFB and a kappa chain reverse amplification PRIMER KR, wherein the KFB is derived from IMGT/prime-DB.

In one realizable approach, the KFB includes the following primers:

KFB1, the nucleotide sequence of which is shown in SEQ ID NO. 9;

KFB2, the nucleotide sequence of which is shown in SEQ ID NO. 10;

KFB3, the nucleotide sequence of which is shown in SEQ ID NO. 11;

KFB4, the nucleotide sequence of which is shown in SEQ ID NO. 12;

KFB5, the nucleotide sequence of which is shown in SEQ ID NO. 13;

KFB6, the nucleotide sequence of which is shown in SEQ ID NO. 14;

KFB7, the nucleotide sequence of which is shown in SEQ ID NO. 15;

KFB8, the nucleotide sequence of which is shown in SEQ ID NO. 16;

KFB9, the nucleotide sequence of which is shown in SEQ ID NO. 17;

KFB10, the nucleotide sequence of which is shown in SEQ ID NO. 18;

KFB11, the nucleotide sequence of which is shown in SEQ ID NO. 19;

KFB12, the nucleotide sequence of which is shown in SEQ ID NO. 20;

KFB13, the nucleotide sequence of which is shown in SEQ ID NO. 21; and

KFB14, the nucleotide sequence of which is shown in SEQ ID NO. 22.

In one achievable mode, the KR has the nucleotide sequence shown in SEQ ID NO. 8.

The present application also aims to provide a composite amplification primer for a lambda strand of a murine monoclonal antibody, wherein the composite amplification primer comprises a forward amplification primer set LFC for a lambda strand and a reverse amplification primer LR for a lambda strand, wherein the LFC comprises:

LFC1, the nucleotide sequence of which is shown in SEQ ID NO. 23; and

LFC2, the nucleotide sequence of which is shown in SEQ ID NO. 24.

Further, the primers in LFC were combined in equal amounts.

In one implementation, the LR nucleotide sequence is set forth in SEQ ID No. 25.

The application also aims to provide a heavy chain amplification composite primer, which comprises a heavy chain forward amplification primer group HFA and a heavy chain reverse amplification primer HR, wherein the HFA is derived from Antibody engineering.

In one implementable manner, the HFA comprises:

HFA1, the nucleotide sequence of which is shown in SEQ ID NO. 26;

HFA2, the nucleotide sequence of which is shown in SEQ ID NO. 27;

HFA3, the nucleotide sequence of which is shown in SEQ ID NO. 28;

HFA4, the nucleotide sequence of which is shown in SEQ ID NO. 29;

HFA5, the nucleotide sequence of which is shown in SEQ ID NO. 30;

HFA6, the nucleotide sequence of which is shown in SEQ ID NO. 31;

HFA7, the nucleotide sequence of which is shown in SEQ ID NO. 32;

HFA8, the nucleotide sequence of which is shown in SEQ ID NO. 33;

HFA9, the nucleotide sequence of which is shown in SEQ ID NO. 34;

HFA10, the nucleotide sequence of which is shown in SEQ ID NO. 35;

HFA11, the nucleotide sequence of which is shown in SEQ ID NO. 36;

HFA12, the nucleotide sequence of which is shown in SEQ ID NO. 37;

HFA13, the nucleotide sequence of which is shown in SEQ ID NO. 38;

HFA14, the nucleotide sequence of which is shown in SEQ ID NO. 39; and

HFA15, the nucleotide sequence of which is shown in SEQ ID NO. 40.

Further, the primers in HFA were combined in equal amounts.

In one achievable approach, the HR is derived from a common gene sequence of mouse IgG1 antibody, IgG2a antibody, IgG2b antibody and IgG3 antibody in the constant region CH1 portion.

Further, the nucleotide sequence of the HR is shown as SEQ ID NO. 41.

The present application also provides an amplification kit comprising the aforementioned primers, specifically, at least one of a kappa strand amplification first composite primer, a kappa strand amplification second composite primer, a lambda strand amplification composite primer, and a heavy chain amplification composite primer.

The present application also provides a method of obtaining a murine single heavy light chain, the method comprising: and (3) respectively amplifying a light chain gene and a heavy chain gene by using a mouse cDNA (complementary deoxyribonucleic acid) as a template and using a mouse monoclonal antibody light chain primer and a mouse monoclonal antibody heavy chain primer, wherein the mouse monoclonal antibody light chain primer is KFA, KFB or LFC as described above, and the mouse monoclonal antibody heavy chain primer is HFA as described above.

Further, the method further comprises, after amplifying the light chain gene and the heavy chain gene:

performing antibody sequencing by using the light chain gene sample and the heavy chain gene sample obtained by amplification;

and searching the genome position of the detected antibody sequence by using a database, and determining the leader peptide sequence of the detected antibody sequence.

Optionally, the database is an IMGT database.

In the present application, the method for determining the leader peptide sequence may be any one of the methods known in the art for determining the leader peptide sequence based on the position of the antibody sequence in the genome.

The present application also provides a method of screening the composite primer, the method comprising:

performing PCR amplification by using a plurality of pairs of candidate primer pairs respectively by using sp2/0cDNA as a template;

and (3) carrying out electrophoretic detection on the PCR amplification result of each pair of candidate primer pairs, and rejecting the candidate primer pairs capable of amplifying the sp2/0cDNA non-functional genes.

In an implementation manner, composite primers are formed according to the electrophoresis detection result, the composite primers comprise a forward primer and a reverse primer with a negative screening result, the forward primer with the negative screening result is a candidate primer pair which cannot amplify the sp2/0cDNA non-functional gene, and the negative screening result means that no amplification product band of the non-functional gene exists in the electrophoresis detection result.

In one implementation, each pair of candidate primers comprises a forward primer and a reverse primer.

In one implementable manner, the plurality of candidate primer pairs are homologous candidate primer pairs.

In one achievable approach, the reverse primers of the homologous candidate primer pairs are identical.

Still further, the candidate PRIMER pairs may be derived from Antibody engineering or IMGT/PRIMER-DB.

Compared with the prior art, the forward primers used by the composite primers provided by the application are all compositions, wherein each group of forward primers used for amplifying the light chain has the primer which can amplify a sp2/0 cell non-functional light chain gene, and the light chain composite primers provided by the application are used for PCR amplification, so that the obtained primers do not need to be TA cloned, but can meet the purity requirement of sequencing.

Drawings

FIG. 1 shows the results of post-PCR electrophoresis using a first set of candidate primer pairs using sp2/0cDNA as a template;

FIG. 2 shows the results of post-PCR electrophoresis using a second set of candidate primer pairs using sp2/0cDNA as a template;

FIG. 3 shows the result of electrophoresis of the PCR product in example 1;

FIG. 4 shows a graph of sequencing peaks of a partial fragment of the VL gene;

FIG. 5 shows a graph of the sequencing peaks of partial fragments of the VH gene.

Detailed Description

Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of methods consistent with certain aspects of the invention, as detailed in the appended claims.

The method for amplifying the heavy and light chain genes of monoclonal antibody IgG in mouse hybridoma and the primers thereof provided by the present application are described in detail by the following specific examples.

In this example, the degenerate bases involved include S: G/C, R: A/G, N: A/T/C/G, M: A/C, Y: C/T, W: A/T, V: G/A/C.

In this example, the applicant believes that a non-functional gene sequence derived from sp2/0, which belongs to the kappa chain of an antibody, is represented by SEQ ID NO.42, and since a frame shift occurs due to a genetic mutation in the chain, the gene sequence cannot be translated into a target product, i.e., a light chain protein product, and instead can serve as a template for PCR to interfere with gene amplification of the target antibody.

In this example, a first set of candidate primer pairs derived from Antibody engineering, wherein the forward primers comprise 12 and the reverse primers comprise 1, each forward primer having the following nucleotide sequence, were used for amplification using sp2/0cDNA as a template:

the nucleotide sequence of K1 is shown in SEQ ID NO. 1;

the nucleotide sequence of K2 is shown in SEQ ID NO. 49;

the nucleotide sequence of K3 is shown in SEQ ID NO. 50;

the nucleotide sequence of K4 is shown in SEQ ID NO. 51;

the nucleotide sequence of K5 is shown in SEQ ID NO. 2;

the nucleotide sequence of K6 is shown in SEQ ID NO. 3;

the nucleotide sequence of K7 is shown in SEQ ID NO. 52;

the nucleotide sequence of K8 is shown in SEQ ID NO. 4;

the nucleotide sequence of K9 is shown in SEQ ID NO. 5;

the nucleotide sequence of K10 is shown in SEQ ID NO. 53;

the nucleotide sequence of K11 is shown in SEQ ID NO. 6;

the nucleotide sequence of K12 is shown in SEQ ID NO. 7.

The nucleotide sequence of the reverse primer KR is shown in SEQ ID NO. 8.

In this example, the KR primer was designed based on the sequence of the kappa chain constant region.

The forward primers are respectively combined with reverse primers to form 12 pairs of candidate primer pairs, and the obtained candidate primer pairs are respectively used for PCR amplification under the following conditions:

step 1: the temperature of the mixture is 95 ℃ for 3 minutes,

step 2: the temperature of the mixture is 95 ℃ for 30 seconds,

step 3: the temperature of the mixture is 55 ℃ for 30 seconds,

step 4: the temperature of the mixture is 72 ℃ for 30 seconds,

step 5: the temperature of the mixture is 72 ℃ for 3 minutes,

step 6: the temperature of the mixture is kept at 10 ℃,

step2 through step4 were cycled 35 times.

The amplification results are subjected to electrophoresis detection, and as shown in fig. 1, as can be seen from fig. 1, the amplification products of the 2 nd, 3 rd, 4 th, 7 th and 10 th primers are all non-functional genes and have false positive interference, and the band in the amplification product of the 11 th primer is impure as a template, and the lane has no band when the PCR is performed again, so that the 2 nd, 3 rd, 4 th, 7 th and 10 th primers are screened out, and the other forward primer groups are the first forward amplification primer group KFA, specifically, the sequence numbers of the forward primers are sequentially shown as SEQ ID No.1 to SEQ ID No. 7.

Specifically, each primer in the first forward amplification primer set KFA was combined in equal amounts.

In this example, a second set of candidate PRIMER pairs from IMGT/prime-DB, wherein the forward PRIMERs comprise 19 and the reverse PRIMERs comprise 1, were used to amplify sp2/0cDNA as a template, and wherein the nucleotide sequence of each forward PRIMER is as follows:

the nucleotide sequence of IMGTK1 is shown in SEQ ID NO. 9;

the nucleotide sequence of IMGTK2 is shown in SEQ ID NO. 54;

the nucleotide sequence of IMGTK3 is shown in SEQ ID NO. 10;

the nucleotide sequence of IMGTK4 is shown in SEQ ID NO. 11;

the nucleotide sequence of IMGTK5 is shown in SEQ ID NO. 55;

the nucleotide sequence of IMGTK6 is shown in SEQ ID NO. 12;

the nucleotide sequence of IMGTK7 is shown in SEQ ID NO. 13;

the nucleotide sequence of IMGTK8 is shown in SEQ ID NO. 14;

the nucleotide sequence of IMGTK9 is shown in SEQ ID NO. 15;

the nucleotide sequence of IMGTK10 is shown in SEQ ID NO. 16;

the nucleotide sequence of IMGTK11 is shown in SEQ ID NO. 17;

the nucleotide sequence of IMGTK12 is shown in SEQ ID NO. 56;

the nucleotide sequence of IMGTK13 is shown in SEQ ID NO. 18;

the nucleotide sequence of IMGTK14 is shown in SEQ ID NO. 19;

the nucleotide sequence of IMGTK15 is shown in SEQ ID NO. 20;

the nucleotide sequence of IMGTK16 is shown in SEQ ID NO. 21;

the nucleotide sequence of IMGTK17 is shown in SEQ ID NO. 22;

the nucleotide sequence of IMGTK18 is shown in SEQ ID NO. 57;

the nucleotide sequence of IMGTK19 is shown in SEQ ID NO. 58.

The nucleotide sequence of the reverse primer KR is shown in SEQ ID NO. 8.

The forward primers are respectively combined with reverse primers to form 19 pairs of candidate primer pairs, and the obtained candidate primer pairs are respectively used for PCR amplification under the following conditions:

step 1: the temperature of the mixture is 95 ℃ for 3 minutes,

step 2: the temperature of the mixture is 95 ℃ for 30 seconds,

step 3: the temperature of the mixture is 55 ℃ for 30 seconds,

step 4: the temperature of the mixture is 72 ℃ for 30 seconds,

step 5: the temperature of the mixture is 72 ℃ for 3 minutes,

step 6: the temperature of the mixture is kept at 10 ℃,

step2 through step4 were cycled 35 times.

The amplification results are subjected to electrophoresis detection, and as shown in fig. 2, as can be seen from fig. 2, the amplification products obtained by the amplification of primers No.2, 5, 12, 18 and 19 are all non-functional genes and have false positive interference, while the amplification products obtained by the amplification of primers No.1 and 9 are impure as templates, and the two long lanes are subjected to PCR again without bands, so that primers No.2, 5, 12, 18 and 19 are screened out, and the rest of the primer sets are a second forward amplification primer set KFB, specifically, the sequences of the primers in the second forward amplification primer set KFB are shown in SEQ ID No.9 to SEQ ID No. 22.

Since the PCR process of the lambda strand is not interfered by the non-functional gene, the sequence numbers of the primers in the lambda strand forward amplification primer set LFC are shown in sequence as SEQ ID NO.23 to SEQ ID NO.24 in this example.

Alternatively, the primers in the LFC are combined in equal amounts.

Further, the lambda chain amplification composite primer also comprises a lambda chain reverse amplification primer LR, and the nucleotide sequence of the LR is shown in SEQ ID No. 25.

The present applicants have found that the lambda strand primer composition can be used for amplification of lambda strands, the number of amplification tubes can be reduced, the lambda strands can be amplified in a single tube, and the resulting lambda strands are pure and can be used directly for sequencing.

Similarly to the PCR process of lamda chain, the PCR process of heavy chain is not interfered by non-functional gene, therefore, in this example, the heavy chain forward amplification primer set HFA is derived from Antibody engineering, specifically, the sequence numbers of the primers are shown as SEQ ID NO.26 to SEQ ID NO.40 in sequence.

In this example, the applicant performed sequence alignment of genes of mouse IgG1, IgG2a, IgG2b, and IgG3 antibody constant region CH1, and selected a region with highly similar sequence to design heavy chain reverse amplification primer HR, wherein the nucleotide sequence table of HR is shown in SEQ ID No. 41.

The applicant has found that by subjecting the heavy chain primer composition to heavy chain amplification, the number of amplification tubes can be reduced, the heavy chain can be amplified in a single tube, and the obtained heavy chain is pure and can be directly used for sequencing.

Examples

Example 1

anti-PCT monoclonal antibody cell strain 2D8, total RNA was extracted, Oligo dT was selected as a primer for reverse transcription to generate cDNA, and cDNA was used as a template for PCR amplification, since Oligo dT specifically binds to poly (A) tail at 3' end of mRNA and only mRNA could be reverse transcribed.

A PCR system was prepared as shown in Table 1 below, with the PCR conditions set as follows:

step 195 ℃ for 3 minutes

step 295 deg.C for 30 seconds

step 355 deg.C for 30 seconds

step 472 deg.C for 30 seconds

step 572 deg.C for 3 minutes

step 610 ℃ hold

step 2-step 4 were cycled 35 times.

TABLE 1

As shown in FIG. 3, the results of PCR according to the above-described method and nucleic acid electrophoresis using a Loading buffer after completion of the reaction were shown in FIG. 3, and it was found that the variable region gene corresponding to the antibody was successfully amplified using both of the kappa light chain and heavy chain combination primers in this example.

Sending each PCR product to a sequencing company for gene sequencing, wherein the 1# PCR tube product and the 2# PCR tube product are VL, and the primer KR is used as a sequencing primer; the product of the 3# PCR tube is VH, the primer HR is used as a sequencing primer, and the sequencing result is shown in the figure and figure 5, wherein figure 4 shows a graph of the sequencing peak shape of the VL gene partial fragment, and figure 5 shows a graph of the sequencing peak shape of the VH gene partial fragment.

As can be seen from FIGS. 4 and 5, the PCR product of the variable region gene amplified by the composite primers used in this example was subjected to gene sequencing after recovery by simple electrophoresis gel, the sequencing peak pattern was clear and single, and there was no interpretation of overlapping peak interference sequences, indicating that the method successfully eliminated the interference effect of the non-functional gene of sp2/0 cells on the sequencing.

The partial nucleotide sequence table of VL is shown in SEQ ID NO.43 because the PCR primers have degenerate bases and the ends of the genes cannot be detected by sequencing.

Further, sequence alignment is carried out by utilizing an IMGT database, and a leader peptide and a partial FR1 region sequence are determined, wherein the nucleotide sequence table of the leader peptide is shown as SEQ ID NO.44, and further the complete VL nucleotide sequence table is determined as shown as SEQ ID NO. 45.

Similarly, the partial nucleotide sequence of VH is shown in SEQ ID NO.46, because the degenerate bases exist in the PCR primers and the ends of the gene cannot be detected by sequencing.

Further, the IMGT database is utilized to carry out sequence comparison, the nucleotide sequence table of the leader peptide is determined to be shown as SEQ ID NO.47, and then the complete VH nucleotide sequence table is determined to be shown as SEQ ID NO. 48.

The present application has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to limit the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the presently disclosed embodiments and implementations thereof without departing from the spirit and scope of the present disclosure, and these fall within the scope of the present disclosure. The protection scope of this application is subject to the appended claims.

Sequence listing

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<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 15

agcctcccta tctgyatcyg 20

<210> 16

<211> 22

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 16

gatgacccag wctcmmaaat tc 22

<210> 17

<211> 21

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 17

tccatggcta taggagaaaa a 21

<210> 18

<211> 21

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 18

ctttccttgc tgtgacagca a 21

<210> 19

<211> 19

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 19

cagccatcct gtctgtgag 19

<210> 20

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 20

gcaytctcca atccwgtcac 20

<210> 21

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 21

gaaccagtct ccatccagtc 20

<210> 22

<211> 21

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 22

gatgacacaa tcttcatcct c 21

<210> 23

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 23

gacgctgttg tgactcagga 20

<210> 24

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 24

gaccytgtgc tcactcagtc 20

<210> 25

<211> 21

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 25

acagactctt ctccacagtg t 21

<210> 26

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 26

gaggttcdsc tgcaacagty 20

<210> 27

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 27

caggtgcaam tgmagsagtc 20

<210> 28

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 28

gavgtgmwgc tggtggagtc 20

<210> 29

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 29

caggttaytc tgaaagagtc 20

<210> 30

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 30

gakgtgcagc ttcagsagtc 20

<210> 31

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 31

gagatccagt tsgygcagtc 20

<210> 32

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 32

cagrtccaac tgcagcagyc 20

<210> 33

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 33

gaggtgmagc tasttgagwc 20

<210> 34

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 34

gaagtgaagm ttgaggagtc 20

<210> 35

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 35

gatgtgaacc tggaagtgtc 20

<210> 36

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 36

cagatkcagc ttmaggagtc 20

<210> 37

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 37

caggcttatc tgcagcagtc 20

<210> 38

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 38

caggttcacc tacaacagtc 20

<210> 39

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 39

caggtgcagc ttgtagagac 20

<210> 40

<211> 19

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 40

gargtgmagc tgktggaga 19

<210> 41

<211> 19

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 41

ttggggggaa gatgaagac 19

<210> 42

<211> 713

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 42

atggagacag acacactcct gttatgggta ctgctgctct gggttccagg ttccactggt 60

gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 120

atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 180

caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 240

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 300

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 360

tcggaggggg gaccaagctg gaaataaaac gggctgatgc tgcaccaact gtatccatct 420

tcccaccatc cagtgagcag ttaacatctg gaggtgcctc agtcgtgtgc ttcttgaaca 480

acttctaccc caaagacatc aatgtcaagt ggaagattga tggcagtgaa cgacaaaatg 540

gcgtcctgaa cagttggact gatcaggaca gcaaagacag cacctacagc atgagcagca 600

ccctcacgtt gaccaaggac gagtatgaac gacataacag ctatacctgt gaggccactc 660

acaagacatc aacttcaccc attgtcaaga gcttcaacag gaatgagtgt tag 713

<210> 43

<211> 320

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 43

ctccactctc cctgcctgtc agtcttggag atcaagcctc cttctcttgc agatctagtc 60

agagccttgt ccacagtaat cgaatcacct atttacattg gtacctgcag aagccaggcc 120

agtctccaaa gctcctgatc tacacagttt ccagccgctt ttctggggtc ccagacaggt 180

tcagtggcag tggatcaggg acagatttca cactcaagat cagcagagtg gaggctgagg 240

atctgggagt ttatttctgc tctcaaagta cacatgttcc gtggacgttc ggtggaggca 300

ccaagctgga aatcaaacgg 320

<210> 44

<211> 57

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 44

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagt 57

<210> 45

<211> 339

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 45

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

ttctcttgca gatctagtca gagccttgtc cacagtaatc gaatcaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acacagtttc cagccgcttt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

tggacgttcg gtggaggcac caagctggaa atcaaacgg 339

<210> 46

<211> 354

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 46

tctggacctg agctggtgaa gcctggggct tcactgaaga tatcctgcaa gacttctgga 60

tacacattca ctgaatacac catgcactgg gtgaagcaga gccatggaaa gagccttgaa 120

tggattggag gtattattcc tgacagtggt ggtactagct acaaccagaa gttcaagggc 180

aaggccacat tgactgtaga caagtcctcc accacagcct acatggagct ccgcagcctg 240

acatctgagg attctgcagt ctattactgt gcaagatatt attactacgg tagtagccct 300

tgttactatg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354

<210> 47

<211> 57

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 47

atggaatgga gctgggtctt tctctttctc ctgtcaggaa ctgcaggtgt cctctct 57

<210> 48

<211> 372

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 48

gaggtccggc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc actgaagata 60

tcctgcaaga cttctggata cacattcact gaatacacca tgcactgggt gaagcagagc 120

catggaaaga gccttgaatg gattggaggt attattcctg acagtggtgg tactagctac 180

aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccac cacagcctac 240

atggagctcc gcagcctgac atctgaggat tctgcagtct attactgtgc aagatattat 300

tactacggta gtagcccttg ttactatgct atggactact ggggtcaagg aacctcagtc 360

accgtctcct ca 372

<210> 49

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 49

gacatccaga tgacacagwc 20

<210> 50

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 50

gatrttgtga tgacccagwc 20

<210> 51

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 51

gacattstgm tgacccagtc 20

<210> 52

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 52

gayattktgc tgactcagtc 20

<210> 53

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 53

gacattgtga tgwcacagtc 20

<210> 54

<211> 21

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 54

ybwgctsacy cartctccwr c 21

<210> 55

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 55

ctgcmtctcy dggggagaag 20

<210> 56

<211> 19

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 56

trtctctrgg gcagagrgc 19

<210> 57

<211> 21

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 57

cccagtctcc atcttatctt g 21

<210> 58

<211> 24

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 58

gacattcagc tgacccagtc tcca 24