METHOD FOR ESTABLISHING EUKARYOTIC EXPRESSION CELL LINE OF CD36 MUTANT GENE THAT ENCODES CD36 DEFICIENCY

Abstract

A method for establishing eukaryotic expression cell line of CD36 mutant gene that encodes CD36 deficiency, the method including: (1) extracting total RNA from a whole blood sample derived from a CD36-deficient individual, and amplifying a coding sequence (CDS) in CD36 mRNA, to obtain a cDNA sequence fragment of the mutant CD36 gene; (2) splicing and amplifying the mutant CD36 gene and the EGFP fluorescent gene by SOE-PCR (Gene Splicing By Overlap Extension PCR) using four forward and reverse primers, to obtain a mutant gene fragment of MT-CD36-EGFP; (3) constructing and amplifying a MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector including the mutant CD36 gene and the EGFP fluorescent gene; (4) transfecting the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector into the CHO-K1 cell line by using virus-mediated transfection of eukaryotic cells.

Claims

1. A method for establishing eukaryotic expression cell line of CD36 mutant gene that encodes CD36 deficiency, the method comprising: (1) extracting total RNA from a whole blood sample derived from a CD36-deficient individual, and amplifying a coding sequence (CDS) in CD36 mRNA by Reverse Transcription PCR (RT-PCR) using a pair of primers, to obtain a cDNA sequence fragment of the mutant CD36 gene, wherein primers for comprise an upstream primer YC-36F having a sequence of SEQ. ID. NO. 1, and a downstream primer YC-36R having a sequence of SEQ. ID. NO. 2; (2) splicing and amplifying the coding sequence (CDS) of the mutant CD36 cDNA (excluding the termination condons) and EGFP fluorescent gene by Gene Splicing by Overlap Extension PCR (SOE-PCR), to obtain a mutant gene fragment of MT-CD36-EGFP comprising a full-length coding sequence (CDS) excluding the terminator of the mutant CD36 cDNA, a full-length EGFP fluorescent gene and desired cleavage sites, wherein primers for SOE-PCR comprises: using a forward primer CD36-F2 and a reverse primer CD36-R3 to amplify a target gene fragment of MT-CD36-EGFP-1 comprising an EcoR 1 cleavage site and protective bases, a full-length coding sequence (CDS) excluding the terminator of CD36 cDNA, a fusion gene BamH I and protective bases, and a portion of the 5-terminal sequence of the EGFP fluorescent reporter gene, wherein the forward primer CD36-F2 also serves as a forward primer for the subsequent splicing and amplification of MT-CD36-EGFP by SOE-PCR, and has a sequence of SEQ. ID. NO. 3, and the reverse primer CD36-R3 has a sequence of SEQ. ID. NO. 4; and using a forward primer EGFP-F4 and a reverse primer EGFP-R2 to amplify a target gene fragment of MT-CD36-EGFP-2 comprising a portion of the 3-terminal fragment of the CD36 CDS cDNA, a fusion gene BamH I and protective bases, a full-length of EGFP CDS fluorescent reporter gene, and an XhoI cleavage site and protective bases, wherein the reverse primer EGFP-R2 also serves as a reverse primer for the subsequent splicing and amplification of MT-CD36-EGFP by SOE-PCR, wherein the forward primer EGFP-F4 has a sequence of SEQ. ID. NO. 5, and the reverse primer EGFP-R2 has a sequence of SEQ. ID. NO. 6; (3) constructing and amplifying a MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector by ligating the MT-CD36-EGFP to a pLV4/StripII-HIS 10 vector; the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector comprising the full-length CDS excluding the terminator of the mutant CD36 cDNA and the EGFP fluorescent gene by ligating MT-CD36-EGFP to a pLV4/StripII-HIS10 vector; (4) transfecting the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector into the CHO-K1 cell line by using virus-mediated transfection of eukaryotic cells, and screening and constructing a eukaryotic cell line MT-CD36-CHO-K1 stablely expressing the mutant CD36 gene that encodes CD36 deficiency; and (5) repeating aforesaid steps, to establish a eukaryotic cell line Normal-CD36-CHO-K1 stably expressing normal CD36 gene as a positive control, and establishing a EGFP-pLV4-CHO-K1 line stably expressing the EGFP fluorescent protein alone as a negative control, wherein in the construction of the negative control EGFP-pLV4-CHO-K1 line, a target gene fragment encoding EGFP comprising an EcoR 1 cleavage site and protective bases, the full-length gene sequence of the EGFP fluorescent reporter gene, and an XhoI cleavage site and protective bases is obtained by amplification using a pair of PRC primers, wherein the PCR primers comprise an upstream primer EGFP-F3 having a sequence of SEQ. ID. NO. 7, and a downstream primer EGFP-R2 that is a reverse primer of the gene fragment of MT-CD36-EGFP-2.

2. The method of claim 1, wherein the upstream primer YC-36F for the RT-PCR amplification of the coding sequence (CDS) of CD36 cDNA is located in a 5-promoter region of the CD36 mRNA sequence, and the downstream primer YC-36R is located in a 3-terminator region of the CD36 mRNA sequence, the amplification region covers the coding sequence (CDS) of CD36 mRNA, and a plurality of mutant CD36 genes with mutations within a linker region of 5-terminal and 3-terminal primer binding regions of the template is obtained through amplification; and for the length of the fragment amplified by using the primers for RT-PCR amplification of the mutant CD36 gene, if the target mutant CD36 gene amplified is a gene with point mutations, the length of the amplified fragment is 1432 bp, and if the target mutant CD36 gene amplified is a mutant gene with base insertions or deletions, the length of the amplified fragment is increased or decreased on the basis of 1432 bp by the base number inserted or deleted.

3. The method of claim 1, wherein the cDNA sequence fragment of the mutant CD36 gene and the EGFP fluorescent gene are spliced and amplified by SOE-PCR, to obtain the target gene fragment of MT-CD36-EGFP comprising the full-length CDS excluding the terminator of the mutant CD36 cDNA, the full-length EGFP fluorescent gene and desired cleavage sites, wherein (1) during the SOE-PCR, the forward primer CD36-F2 for obtaining the target gene fragment of MT-CD36-EGFP-1 is located in a 5-promoter region of the cDNA sequence fragment of the mutant CD3 6 gene; the target gene fragment of MT-CD36-EGFP-1 comprises an EcoR 1 cleavage site and protective bases, a full-length CDS excluding the terminator of the CD36 cDNA, a fusion gene BamH I and protective bases, and a portion of the 5-terminal sequence of the EGFP fluorescent reporter gene, the EcoR 1 cleavage site and protective bases are comprised at a 5 terminus of the primer, and the forward primer CD36-F2 also serves as the forward primer for the subsequent splicing and amplification of MT-CD36-EGFP by SOE-PCR; the reverse primer CD36-R3 is located in a 3-terminal region excluding the terminator of the CD36 cDNA sequence fragment of the mutant CD36 gene, and the portion of the 5-terminal promoter region sequence of the EGFP fluorescent reporter gene and the fusion gene BamH I and protective bases are comprised at the 5 terminus of the reverse primer CD36-R3; and for the length of the target gene fragment of MT-CD36-EGFP-1, when the mutant CD36 gene comprise point mutations, the length of the target gene fragment of MT-CD36-EGFP-1 is 1441 bp, and when the mutant CD36 gene comprise base insertions or deletions, the length of the target gene fragment of MT-CD36-EGFP-1 is increased or decreased on the basis of 1441 bp by the base number inserted or deleted; (2) during the SOE-PCR, the forward primer EGFP-F4 for obtaining the target gene fragment of MT-CD36-EGFP-2 is located in a 5-promoter region of the sequence of the EGFP fluorescent reporter gene; the target gene fragment of MT-CD36-EGFP-2 comprises a portion of the 3-terminal fragment of the CD36 coding cDNA region, a fusion gene BamH I and protective bases, a full-length EGFP fluorescent reporter gene, and an XhoI cleavage site and protective bases, a 3-terminal 10-base sequence before the terminator TAA of the CD36 cDNA CDS sequence and the fusion gene BamH I and protection bases are comprised at the 5 terminus of the forward primer EGFP-F4; the reverse primer EGFP-R2 is located in the 3-terminator region of the EGFP fluorescent gene, and the XhoI cleavage site and protective bases are comprised at a 5 terminus of the reverse primer EGFP-R2; and the length of the target gene fragment of MT-CD36-EGFP-2 is 753 bp; and (3) by using the forward primer CD36-F2 and the reverse primer EGFP-R2, and using MT-CD36-EGFP-1 and MT-CD36-EGFP-2 obtained through PCR amplification as templates, the gene fragment of MT-CD36-EGFP is obtained by splicing and amplification by SOE-PCR; the fragment of MT-CD36-EGFP comprises the full-length CDS excluding the terminator of the mutant CD36 gene, a fusion gene BamH I and protective bases the full-length fragment CDS of the EGFP fluorescent gene, and desired EcoR1 and XhoI cleavage sites and for the length of MT-CD36-EGFP, when the mutant CD36 gene comprise point mutations, the length of the amplified fragment of MT-CD36-EGFP is 2165 bp, and when the mutant CD36 gene comprise base insertions or deletions, the length of the amplified fragment of MT-CD36-EGFP is increased or decreased on the basis of 2165 bp by the base number inserted or deleted.

4. The method of claim 1, wherein during the construction of the negative control EGFP-pLV4-CHO-K1 line, the upstream primer EGFP-F3 for PCR amplification to obtain the target gene fragment encoding EGFP is located in a 5-promoter region of the sequence of the EGFP fluorescent gene; the target gene fragment encoding EGFP comprises a EcoR 1 cleavage site and protective bases, the full-length gene sequence of the CDS of the EGFP fluorescent reporter gene, and a XhoI cleavage site and protective bases, and the EcoR 1 cleavage site and protective bases are further comprised at a 5 terminus of the upstream primer EGFP-F3; the downstream primer EGFP-R2 are also the reverse primer for amplification of the gene fragment of MT-CD36-EGFP-2; and the length of the target gene fragment encoding EGFP by PCR is 743 bp.

5. The method of claim 2, wherein the RT-PCR amplification using one pair of primers for RT-PCR is carried out in a PCR machine using the TaKaRa One step RNA PCR Kit (AMV), through a process which is performed under the following cycling parameters: TABLE-US-00031 50 C. 30 min 94 C. 2 min; 30 amplification cycles of 94 C. 30 sec 60 C. 30 sec 72 C. 2 min; and extension and storage at 72 C. 10 min 12 C. .

6. The method of claim 3, wherein the reaction system for PCR amplification of the target fragment of MT-CD36-EGFP-1 has a composition of TABLE-US-00032 ddH.sub.2O 15 L 5x SF Buffer avalaible from Vazyme, 5 L with 10 mM MgCl.sub.2 dNTP mix, 10 mM each 0.5 L CD36-F2, 10 M 1 L CD36-R3, 10 M 1 L MT-CD36 cDNA 1 L DMSO 1 L HS suffer-Fieviy DNA polymerase 0.5 L; from Vazyme the reaction system for PCR amplification of the target fragment of MT-CD36-EGFP-2 has a composition of TABLE-US-00033 ddH.sub.2O 16 L 5x SF Buffer from Vazyme, with 10 mM MgCl.sub.2 5 L dNTP mix, 10 mM each 0.5 L EGFP-F4, 10 M 0.5 L EGFP-R2, 10 M 0.5 L pEGFP-N1 plasmid 1 L DMSO 1 L HS suffer-Fieviy DNA polymerase from Vazyme 0.5 L.

7. The method of claim 6, wherein the PCR amplification using two pairs of PCR primers is carried out in a PCR machine through a process which is performed under the following cycling parameters: TABLE-US-00034 96 C. 5 min 33 amplification cycles of: 96 C. 30 sec 60 C. 30 sec 72 C. 90 sec; and extension and storage at 72 C. 10 min 12 C. .

8. The method of claim 3, wherein the reaction system for PCR amplification to obtain the target fragment of MT-CD36-EGFP by SOE-PCR has a composition of TABLE-US-00035 H.sub.2O 24 L phanta 5x SF Buffer, with 10 mM Mgcl2 10 L dNTP Mix, 10 mM each 1 L MT-CD36-EGFP-1 4 L MT-CD36-EGFP-2 4 L phanta HS Suffer-Fideliy DNA Polymerase 1 L; the amplification is carried out in a PCR machine through a process which is performed under the following cycling parameters: TABLE-US-00036 98 C. 5 min; and 15 amplification cycles of 98 C. 5 min 72 C. 1 min a system having the following composition is added to the reaction tube after the reaction is completed: TABLE-US-00037 CD36-F2, 10 M 2 L EGFP-R2, 10 M 2 L DMSO 2 L; and amplification is continued through a process which is performed under the following cycling parameters: TABLE-US-00038 20 amplification cycles of 98 C. 10 sec 56 C. 30 sec 72 C. 60 sec; and extension and storage at: 72 C. 10 min 12 C. .

9. The method of claim 4, wherein the reaction system for PCR amplification to obtain the target fragment of the EGFP gene comprising a EcoR 1 cleavage site and protective bases, the full-length CDS of EFGP flurescent gene, and a XhoI cleavage site and protective bases has the same composition as that of the reaction system for PCR amplification to obtain the target gene fragment of MT-CD36-EGFP-2, except that the upstream primer is EGFP-F3; and the cycling parameters for amplification in a PCR machine under the following cycling parameters: TABLE-US-00039 96 C. 5 min 33 amplification cycles of: 96 C. 30 sec 60 C. 30 sec 72 C. 90 sec; and extension and storage at 72 C. 10 min 12 C. .

10. The method of claim 1, wherein a process for constructing and amplifying the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector by ligating MT-CD36-EGFP to the pLV4/StripII-HIS10 vector comprises: (1) ligating the enzymatically cleaved products MT-CD36-EGFP and pLV4/StripII-HIS10 vector by using the T4 DNA Ligase Kit (Promega), of that the enzymatically cleaved products have been cleaved by using the EcoR1/XhoI DNA Endonuclease Kit (Beyotime Biotechnology) and than have been purified, to obtain the pLV4/StripII-HIS10 expression vector MT-CD36-EGFP-pLV4/StripII-HIS 10 comprising the target fragment of the mutant CD36 gene and the EGFP fluorescent gene, wherein the reaction system for ligation comprises: TABLE-US-00040 MT-CD36-EGFP:eukaryotic vector pLV4/StripII-HIS10 molar ratio 7:1, total volume: 1-8 L T4 DAN ligase 10x Buffer 1 L T4 DAN ligase 1 L H.sub.2O q.s. to 10 L; and (2) transforming the ligated product MT-CD3 6-EGFP-pLV4/StripII-HIS10 into DH5 Chemically Competent Cells (Beijing TransGen Biotech, Inc) under the temperature conditions comprising: TABLE-US-00041 ice bath 30 min; heat shock at 42 C. 1 min; and ice bath 2 min.

11. The method of claim 10, wherein the colony of the DH5 Chemically Competent Cells successfully transformed with the MT-CD36-EGFP-pLV4/StripII-HIS10 is confirmed by colony PCR by amplifying the gene fragment of MT-CD36-EGFP in the MT-CD36-EGFP-pLV4/StripII-HIS10 plasmid in the transfected colony using the upstream primer CD36-F2 and the downstream primer EGFP-R2 for SOE-PCR, wherein the colony PCR is carried out using 2Taq Master Mix Kit (Vazyme), and the colony is suspended and lyzed by incubating for 20 min at 100 C. before the colony PCR, where the reaction system for colony PCR comprises: TABLE-US-00042 H.sub.2O 0.5 L 2 Taq Master Mix 12.5 L upstream primer CD36-F2, 10 M 1 L downstream primer EGFP-R2, 10 M 1 L bacterial suspension 10 L.

12. The method of claim 11, wherein the colony PCR using one pair of primers is carried out in a PCR machine through a process which is performed under the following cycling parameters: TABLE-US-00043 98 C. 5 min 33 amplification cycles of: 98 C. 10 sec 56 C. 30 sec 72 C. 90 sec; and extension and storage at: 72 C. 10 min 12 C. .

13. The method of claim 1, wherein transfecting the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector into the CHO-K1 cell line by using virus-mediated transfection of eukaryotic cells, and screening and constructing a eukaryotic cell line MT-CD36-CHO-K1 stablely expressing the mutant CD36 gene comprises: (1) subjecting the 293-T cell line in logarithmic growth phase to lentiviral packaging by using the Lenti-Pac HIV Expression packaging Kit (GeneCopoeia) to obtain a virus suspension, wherein 2 hrs before the 293-T cell line is transfected by lentiviral packaging, the cell culture medium of the 293-T cell line is changed to a serum-free medium; and (2) infecting the CHO-K1 cell line with the virus suspension obtained after lentiviral packaging, and screening the CHO-K1 cell line infected with the virus suspension using puromycin (Sigma), to establish a eukaryotic cell line MT-CD36-CHO-K1 stablely expressing the mutant CD36 gene, wherein the concentration of puromycin used for screening is 0.2 g/mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1 is a schematic view showing the primers for amplification of a target fragment of CD36-EGFP-1 by SOE-PCR.

[0065] FIG. 2 is a schematic view showing the primers for amplification of a target fragment of CD36-EGFP-2 by SOE-PCR.

[0066] FIG. 3 is a schematic view showing the final target fragment of MT-CD36-EGFP amplified by SOE-PCR.

[0067] FIG. 4 shows the exon sequencing result of the CD36 gene in a CD36-deficient individual ZYT in Example 1, showing that the exon-4 has C220T mutation.

[0068] FIG. 5 shows amplification of the CD36 cDNA from the CD36-deficient individual ZYT (1), the target fragments of ZYT-CD36-EGFP-1 (2) and ZYT-CD36-EGFP-2 (3), and the product ZYT-CD36-EGFP (4) ligated by SOE-PCR in Example 1.

[0069] FIG. 6 shows the cDNA sequencing result of the CD36 gene in a CD36-deficient individual ZYT in Example 1, showing 220C>T (Gln74Stop) mutation.

[0070] FIG. 7 shows the sequencing result of the 220T-CD36-EGFP-pLV4/StripII-HIS 10 eukaryotic expression vector successfully constructed in Example 1.

[0071] FIG. 8 shows the cDNA sequencing result of the eukaryotic CHO-K1 cell line (220T-CD36-CHO-K1 line) stably expressing the mutant CD36 gene (220C>T) in Example 1.

[0072] FIG. 9 shows the results of confirming CD36 protein expression in the eukaryotic CHO-K1 cell line (220T-CD36-CHO-K1 line) stably expressing the mutant CD36 gene (220C>T) by flow cytometry in Example 1, in which (1): the 220T-CD36-CHO-K1 line is negative in CD36 protein expression; (2): the negative control EGFP-pLV4-CHO-K1 line is negative in CD36 protein expression; (3) the blank control CHO-K1 line CD36 is negative in CD36 protein expression; and (4): the positive control NORMAL-CD36-CHO-K1 line is positive in CD36 protein expression.

[0073] FIG. 10 shows the results of confirming CD36 protein expression in the eukaryotic CHO-K1 cell line (220T-CD36-CHO-K1 line) stably expressing the mutant CD36 gene (220C>T) by western-blotting in Example 1, in which (1): the 220T-CD36-CHO-K1 line is negative in CD36 protein expression; (2): the negative control EGFP-pLV4-CHO-K1 line is negative in CD36 protein expression; (3) the blank control CHO-K1 line is negative in CD36 protein expression; and (4): the positive control NORMAL-CD36-CHO-K1 line is positive in CD36 protein expression.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0074] The following examples are further explanations and illustrations of the disclosure, and not intended to limit the disclosure in any way.

[0075] On the basis of molecular genetics leading to CD36 deficiency, the disclosure provides a method for establishing eukaryotic stable expression cell line of CD36 mutant gene that encodes human CD36 deficiency. The method for establishing eukaryotic stable expression cell line of CD36 mutant gene that encodes CD36 deficiency can be established by designing primers for RT-PCR of mutant CD36 gene and primers for construction of a eukaryotic vector, exploring the optimal annealing temperature and adjusting the conditions comprising Mg.sup.2+ concentration and primer concentration, exploring the optimal enzymatic cleavage conditions in the construction of the eukaryotic vector for the mutant CD36 gene and the reaction conditions for ligating a insert fragment to the eukaryotic vector, and exploring the viral packaging, transfection, and screening conditions for establishing the eukaryotic cell line stably expressing the mutant CD36 gene by virus-mediated transfection.

Example 1

[0076] In this example, an implementation for establishing a eukaryotic cell line, 220T-CD36-CHO-K1 line stably expressing the mutant CD36 gene 220C>T(Gln74stop) (GenBank Accession No.: KF539919.1) encoding CD36 deficiency by the method for establishing eukaryotic stable expression cell line of CD36 mutant gene that encodes CD36 deficiency according to the disclosure is specifically described.

[0077] A CD36-deficient individual that was confirmed to have mutation of C220T by exon sequencing of the CD36 gene (where the individual was designated as ZYT, and the exon sequencing results were shown in FIG. 4) was chosen. 5 mL of venous blood was drawn from the individual with informed consent, EDTA was added for anti-coagulation, and total RNA was extracted from the blood sample. A target fragment of CD36 cDNA (ZYT-CD36 cDNA) was amplified by RT-PCR using the first pair of PCR primers of the disclosure comprising an upstream primer YC-36F and a downstream primer YC-36R, and using the TaKaRa One step RNA PCR Kit (AMV). The amplification was carried out in ABI 9700PCR machine, and the reaction system for PCR amplification comprised:

TABLE-US-00015 10 one step RNA PCR Buffer 5 L 25 mM MgCl2 10 L 10 mM dNTPs 5 L Rnase Inhibitor (40 u/L) 1 L AMV Rtase XL 1 L AMV-Optimized Taq 1 L Primer YC-36F (20 M) 1 L Primer YC-36R (20 M) 1 L RNA (100-500 ng/L) 10 L H.sub.2O 15 L

[0078] The cycling parameters comprised:

TABLE-US-00016 50 C. 30 min 94 C. 2 min 30 amplification cycles of: 94 C. 30 sec 60 C. 30 sec 72 C. 2 min; and extension and storage at: 72 C. 10 min 12 C. .

[0079] 5 L of the PCR product was stained by DNA green fluorescent dye (Beijing Tiandz Gene Technology Co., Ltd), and subjected to 1.5% agarose gel electrophoresis. With DL2000 Plus DNA Marker from Vazyme as a control, a band of specific PCR product was observed in a gel imaging system. The band of the PCR product was clear and specific, as shown in FIG. 5(1). 5 L of the PCR product was sequenced, and the sequencing results were shown in FIG. 6. The remaining PCR product was stored at 80 C. until use.

[0080] Next, a target gene fragment of ZYT-CD36-EGFP-1 comprising an EcoR 1 cleavage site and protective bases, a full-length gene sequence of the CD36 protein coding region, a fusion gene BamH I and protective bases, and a portion of the 5-terminal sequence of the EGFP fluorescent reporter gene was obtained by amplification using the second pair of PCR primers of the present invention comprising an upstream primer CD36-F2 and a downstream primer CD36-R3 and using ZYT-CD36 cDNA as a DNA template. The amplification was carried out in ABI 9700 PCR machine, and the reaction system for PCR amplification comprised a composition of:

TABLE-US-00017 ddH.sub.2O 15 L 5 SF Buffer (with 10 mM MgCl2) (Vazyme) 5 L dNTP mix (10 mM each) 0.5 L CD36-F2 (10 M) 1 L CD36-R3 (10 M) 1 L ZYT-CD36 cDNA 1 L DMSO 1 L HS suffer-Fieviy DNA polymerase (Vazyme) 0.5 L

[0081] The cycling parameters comprised:

TABLE-US-00018 96 C. 5 min 33 amplification cycles of: 96 C. 30 sec 60 C. 30 sec 72 C. 90 sec; and extension and storage at: 72 C. 10 min 12 C. .

[0082] 5 L of the PCR product was stained by DNA green fluorescent dye (Beijing Tiandz Gene Technology Co., Ltd), and subjected to 1.5% agarose gel electrophoresis.

[0083] With DL2000 Plus DNA Marker from Vazyme as a control, a band of specific PCR product was observed in a gel imaging system. The band of the PCR product was clear and specific, as shown in FIG. 5(2). The remaining PCR product was stored at 80 C. until use.

[0084] A target gene fragment of ZYT-CD36-EGFP-2 comprising a portion of the 3-terminal fragment of the CD36 encoding cDNA region, a fusion gene BamH I and protective bases, the full-length gene sequence of the EGFP fluorescent reporter gene, and a XhoI cleavage site and protective bases was obtained by amplification using the third pair of PCR primers of the disclosure comprising an upstream primer EGFP-F4 and a downstream primer EGFP-R2 and using the eukaryotic plasmid pEGFP-N1 containing the EGFP fluorescent reporter gene (manufactured by Suzhou Genepharma Co., Ltd) as a template. The amplification was carried out in ABI 9700 PCR machine, and the reaction system for PCR amplification comprised a composition of:

TABLE-US-00019 ddH.sub.2O 16 L 5 SF Buffer (with 10 mM MgCl2) (Vazyme) 5 L dNTP mix (10 mM each) 0.5 L EGFP-F4 (10 M) 0.5 L EGFP-R2 (10 M) 0.5 L pEGFP-N1 plasmid 1 L DMSO 1 L HS suffer-Fieviy DNA polymerase (Vazyme) 0.5 L

[0085] The cycling parameters comprised:

TABLE-US-00020 96 C. 5 min 33 amplification cycles of: 96 C. 30 sec 60 C. 30 sec 72 C. 90 sec; and extension and storage at: 72 C. 10 min 12 C.

[0086] 5 L of the PCR product was stained by DNA green fluorescent dye (Beijing Tiandz Gene Technology Co., Ltd), and subjected to 1.5% agarose gel electrophoresis.

[0087] With DL2000 Plus DNAMarker from Vazyme as a control, a band of specific PCR product was observed in a gel imaging system. The band of the PCR product was clear and specific, as shown in FIG. 5(3). The remaining PCR product was stored at 80 C. until use.

[0088] A gene fragment of ZYT-CD36-EGFP comprising the full-length cDNA encoding region of the mutant CD36 gene ZYT, a fusion gene BamH I and protective bases, the full-length EGFP fluorescent gene, and desired EcoR1 and XhoI cleavage site was obtained by splicing and amplication by SOE-PCR using the upstream primer CD36-F2 in the second pair of primers and the downstream primer EGFP-R2 in the third pair of primers of the disclosure, and using ZYT-CD36-EGFP-1 and ZYT-CD36-EGFP-2 as templates. The amplification was carried out in ABI 9700 PCR machine, and the reaction system for PCR amplification comprised a composition of:

TABLE-US-00021 H.sub.2O 24 L 5 SF Buffer (with 10 mM MgCl2) (phanta) 10 L dNTP Mix (10 mM each) 1 L ZYT-CD36-EGFP-1 4 L ZYT-CD36-EGFP-2 4 L phantaHS Suffer-Fideliy DNA Polymerase 1 L

[0089] The cycling parameters comprised:

TABLE-US-00022 98 C. 5 min

[0090] 15 amplification cycles of:

TABLE-US-00023 98 C. 5 min 72 C. 1 min.

[0091] A system having the following composition was added to the reaction tube after the reaction was completed:

TABLE-US-00024 CD36-F2 2 L EGFP-R2 2 L DMSO 2 L.

[0092] Amplification is continued through a process which is performed under the following cycling parameters:

TABLE-US-00025 20 amplification cycles of: 98 C. 10 sec 56 C. 30 sec 72 C. 60 sec; and extension and storage at: 72 C. 10 min 12 C. .

[0093] 5 L of the PCR product was stained by DNA green fluorescent dye (Beijing Tiandz Gene Technology Co., Ltd), and subjected to 1.5% agarose gel electrophoresis. With DL2000 Plus DNAMarker from Vazyme as a control, a band of specific PCR product was observed in a gel imaging system. The band of the PCR product was clear and specific, as shown in FIG. 5(4). The target fragment of 2165 bp was taken by gel cutting, and subjected to gel extraction by using the AxyPrep DNA Gel Extraction Kit (AXYGEN). 4 L of the extracted product was sequenced, to confirm that the amplified product is consistent in sequence with the target fragment. Then, the remaining extracted product was stored at 80 C. until use.

[0094] After the purified target fragment of ZYT-CD36-EGFP was obtained, ZYT-CD36-EGFP and the eukaryotic vector pLV4/StripII-HIS10 (Shenzhen Angran Biotechnology Co., Ltd.) were enzymatically cleaved by using the EcoR1/XhoI DNA Endonuclease Kit (Beyotime Biotechnology). The reaction system comprised:

TABLE-US-00026 ZYT-CD36-EGFP or eukaryotic 14 L vector pLV4/StripII-HIS10 10 Buffer Y 2 L ECOR 1 1 L Xhol 1 L ddH.sub.2O 2 L

[0095] The reaction condition for enzymatic cleavage comprised: incubation at 37 C. for 1 hr.

[0096] After enzymatic cleavage, the enzymatically cleaved product was purified through the following process.

[0097] Anhydrous ethanol that was 2-3 times the volume of the enzymatically cleaved product and pre-frozen at 80 C. was added to the enzymatically cleaved product, stood at 20 C. for 20 min, and centrifuged at 4 C. for 10 min. The supernatant was removed and, and the ethanol was air dried (for 5-8 min). 12 L of TE Buffer (elution buffer) was added, and stood for 8 min. The concentration of MT-CD36-EGFP and the eukaryotic vector pLV4/StripII-HIS10 was determined respectively and then stored at 20 C. for later use.

[0098] The purified enzymatically cleaved products of MT-CD36-EGFP and the eukaryotic vector pLV4/StripII-HIS10 were ligated by using the T4 DNA Ligase Kit (Promega), to obtain a pLV4/StripII-HIS10 expression vector (C220T-CD36-EGFP-pLV4/StripII-HIS10) comprising the target fragment of the mutant CD36 gene and the EGFP fluorescent gene. The reaction system for ligation comprised:

TABLE-US-00027 ZYT-CD36-EGFP: eukaryotic vectorpLV4/StripII-HIS10 molar ratio 7:1 (total volume: 1-8 L) T4 DAN ligase 10 Buffer 1 L T4 DAN ligase 1 L H.sub.2O q.s. to 10 L.

[0099] Reaction condition for ligation comprised: incubation overnight at 4 C.

[0100] The ligated product was transformed into DH5a Chemically Competent Cells (Beijing TransGen Biotech, Inc). Positive clones were picked up from the E. coli DH5a cell line, cultured, multiplied, and confirmed, to obtain a successfully constructed 220T-CD36-EGFP-pLV4/StripII-HIS10eukaryotic expression vector. The process was specifically as follows.

[0101] The DH5 Chemically Competent Cells (Beijing TransGen Biotech, Inc) were removed from a freezer at 80 C., and thawed by standing on ice for 5 min. 50 L of the DH5a Chemically Competent Cells was aspirated to 10 L of the ligated product obtained in the previous step (1 L of a solution III was added to the ligated product before transformation), stood in an ice bath for 30 min, heat shocked at 42 C. for 1 min, and then stood in an ice bath for 2 min. 1 mL of the SOC medium was then added, and shaken for 1 hr on a shaker at 180 rpm. 300 L of the bacterial suspension was aspirated and inoculated in a plate (a plate with LB medium to which Ampicillin (Amp, final concentration: 100 g/mL) was added). The plate was transferred to an incubator and incubated overnight at 37 C. A single colony was picked up from the plate with well-grown transformants and transferred to a 1.5 mL centrifuge tube (to which 1 mL of Amp-containing SOC medium was added). The bamboo sticks for picking up the bacteria were correspondingly transferred one by one to 0.2 mL centrifuge tubes (to which 12 L sterilized ddH.sub.2O was added), and labeled. The 1.5 mL centrifuge tube containing the bacterial suspension was shaken for 7-8 hrs on a shaker at 37 C.

[0102] During the culture and multiplication process, colony PCR amplification was carried out by using the 2Taq Master Mix Kit (Vazyme) using the upstream primer CD36-F2 in the second pair of primers and the downstream primer EGFP-R2 in the third pair of primers of the present disclosure and using the bacterial suspension in the 0.2 mL centrifuge tube (which was lyzed by incubating for 20 min at 100 C. before PCR amplification) as a template. The amplification was carried out in ABI 9700 PCR machine, and the reaction system for PCR amplification comprised a composition of:

TABLE-US-00028 H.sub.2O 0.5 L 2 Taq Master Mix 12.5 L upstream primer CD36-F2 (10 M) 1 L downstream primer EGFP-R2 (10 M) 1 L bacterial suspension 10 L

[0103] The cycling parameters comprised:

TABLE-US-00029 98 C. 5 min 33 amplification cycles of: 98 C. 10 sec 56 C. 30 sec 72 C. 90 sec; and extension and storage at: 72 C. 10 min 12 C. .

[0104] 5 L of the PCR product was stained by DNA green fluorescent dye (Beijing Tiandz Gene Technology Co., Ltd), and subjected to 1.5% agarose gel electrophoresis. With DL2000 Plus DNAMarker from Vazyme as a control, a band of specific PCR product was observed in a gel imaging system. The band of the PCR product of the picked colony was clear and specific, and the size of the fragment was in agreement with the size of the product ZYT-CD36-EGFP amplified by SOE-PCR. The corresponding colony was multiplied and cultured, and a part was aspirated for validation by sequencing and the remaining is preserved for later use. The result of confirmation by sequencing indicates CD36 220T (as shown in FIG. 7). 100-200 L of the bacterial suspension transformed with a clone in which the remaining sequence is consistent with that in the ligated sequence of ZYT-CD36-EGFP was added to 16 mL LB medium, shaken overnight on a shaker at 37 C., multiplied and cultured to obtain a successfully constructed 220T-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector. The plasmid was extracted by using an endotoxin-free Plasmid mini Preparation Kit (Tiangen Biotech Co, Ltd.). 5 L of the extracted plasmid was subjected to 1.5% agarose gel electrophoresis, to observe that the size of the band is proper. The remaining plasmid was stored at 20 C. for later use.

[0105] Finally, the 220T-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector was transfected into the CHO-K1 cell line by using virus-mediated transfection of eukaryotic cells, and screened to construct a eukaryotic cell line (220T-CD36-CHO-K1) stably expressing the mutant CD36 220C>T gene encoding CD36 deficiency. The process was specifically as follows.

[0106] Lentiviral packaging by using the Lenti-Pac HIV Expression packaging Kit (GeneCopoeia): 210.sup.8 cells of the 293-T cell line in logarithmic growth phase were passaged and inoculated into a 25 mL cell culture flask, cultured for 24 hrs, and transfected when the cells reached 70%-80% confluence. 2 hrs before transfection, the cell culture medium was changed to Opti-MEM I medium (GIBCO). 200 L of a lentiviral packaging plasmid mix was formulated (in a sterilized 1.5 mL centrifuge tube, where 5 g of the eukaryotic expression plasmid 220T-CD36-EGFP-pLV4/StripII-HIS10 successfully constructed and 5 L of the LentiPac HIV reagent were added, and diluted with Opti-MEM I medium to a final volume of 200 L). 200 L of a solution containing EndoFectin transfection reagent was formulated in another sterilized 1.5 mL centrifuge tube by adding 185 L Opti-MEM I medium and 15 L EndoFectin transfection reagent. The formulated solution of the EndoFectin transfection reagent was slowly added dropwise to the lentiviral packaging plasmid mix with gently shaking to mix them uniformly. Then, the solution was incubated for 10-25 min at room temperature, to produce a DNA-EndoFectin mix. The DNA-EndoFectin mix was transferred to a culture of 293T cells, mixed uniformly, and incubated for 8 hrs in a cell incubator at 5% CO.sub.2 and 37 C. The medium containing the transfection mix was removed, and 5 mL of DMEM medium containing 10% fetal bovine serum was added to each flask of cells. 10 L of TiterBoost reagent was further added, and the culture flask was gently shaken, to mix the medium and the reagent uniformly, and then incubated in a cell incubator at 37 C. and 5% CO.sub.2.

[0107] Harvest of the virus suspension: The culture supernatants of 293T cells were collected 48 hours and 72 hours after transfection (after the virus suspension was collected at 48 hrs, 5 mL of DMEM medium containing 10% fetal bovine serum and 10 L of TiterBoost reagent were added to the culture flask), and centrifuged at 4000 g for 10 min at 4 C. to remove cell debris. The supernatant was filtered through a 0.45 M filter into a 15 mL centrifuge tube, biologically measured for the virus titer and stored at 80 C. until use.

[0108] Establishment of a eukaryotic cell line (220T-CD36-CHO-K1 line) stably expressing the mutant CD36 220C>T gene by transfecting the CHO-K1 cell line using the harvested virus suspension, and screening with puromycin: 410.sup.7 CHO-K1 cells in logarithmic growth phase were inoculated into a 25 mL culture flask, added with DMEM medium containing 10% fetal bovine serum and 1% penicillinstreptomycin solution, and cultured for 24 hrs. The medium was removed when the cells reached 80%-90% confluence, and 4 mL of DMEM medium (containing 10% heat-inactivated fetal bovine serum and 1% penicillinstreptomycin solution) and 100 L of the viral suspension (with a virus titer of 210.sup.5 TU/mL) were added, and cultured overnight at 5% CO.sub.2 and 37 C. The medium was removed, and the cells were screened by using 5 mL of DMEM medium containing 0.2 g/mL puromycin (Sigma) (containing 10% heat-inactivated fetal bovine serum and 1% penicillinstreptomycin solution). The state of cells was observed every 24 hours. The medium containing puromycin was replaced every 48 hours and the culture was continued for at least one month.

[0109] A Normal-CD36-CHO-K1 line stably expressing normal CD36 and a EGFP-pLV4-CHO-K1 line having merely EGFP fluorescent gene-containing plasmid transformed therein and stably expressing the EGFP fluorescent protein alone were established as a positive control and a negative control respectively in experiment. The process was as follows.

[0110] Total RNA was extracted from a whole blood sample derived from a CD36 expression-positive individual, and following the process in the above steps, a Normal-CD36-CHO-K1 line stably expressing normal CD36 gene was established as a positive control in an experiment.

[0111] A EGFP-pLV4-CHO-K1 line having EGFP fluorescent gene-containing plasmid transformed therein and stably expressing the EGFP fluorescent protein alone was established as a negative control. The experimental method was as follows.

[0112] PCR amplification was carried out by using the eukaryotic plasmid pEGFP-N1 containing the EGFP fluorescent reporter gene (manufactured by Suzhou Genepharma Co., Ltd) as a template and using EGFP-F3 and EGFP-R2 as forward and reverse primer, to obtain a target EGFP gene fragment comprising an EcoR 1 cleavage site and protective bases, the full-length gene sequence of the EGFP fluorescent reporter gene, and a XhoI cleavage site and protective bases. The reaction system had a composition of (the total volume of the reaction system was 25 L):

TABLE-US-00030 ddH.sub.2O 16 L 5 SF Buffer (with 10 mM Mgcl2) (Vazyme) 5 L dNTP mix (10 mM each) 0.5 L EGFP-F3 (10 M) 0.5 L EGFP-R2 (10 M) 0.5 L pEGFP-N1 plasmid 1 L DMSO 1 L HS suffer-Fieviy DNA polymerase (Vazyme) 0.5 L

[0113] The cycling parameters for PCR amplification were the same as those for amplification of the target fragments of ZYT-CD36-EGFP-1 and ZYT-CD36-EGFP-2 in the above steps.

[0114] The amplified EGFP gene product was subjected to 1.5% agarose gel electrophoresis (105 v, 40 min), gel cutting, and gel extraction by using the AxyPrep DNA Gel Extraction Kit (AXYGEN). The extracted product was sequenced, to confirm that the amplified product is consistent in sequence with the target fragment. Following the method for constructing the 220T-CD36-EGFP-pLV4/StripH-HIS10 plasmid and the method for establishing and screening the 220T-CD36-CHO-K1 cell line, a eukaryotic expression vector for expressing the EGFP gene was constructed, and an EGFP-pLV4-CHO-K1 line stably expressing the EGFP fluorescent protein alone was established.

[0115] Confirmation of the established 220T-CD36-CHO-K1 line:

[0116] RNA was respectively extracted from the 220T-CD36-CHO-K1 line and the positive control Normal-CD36-CHO-K1 line in logarithmic growth phase. A target fragment of CD36 cDNA was amplified by RT-PCR using the first pair of PCR primers comprising the upstream primer YC-36F and the downstream primer YC-36R and using TaKaRa One step RNA PCR Kit (AMV). The amplification was carried out in ABI 9700 PCR machine, and the reaction system for amplification and the cycling parameters were as described in the step 1. 5 L of the PCR product was stained by DNA green fluorescent dye (Beijing Tiandz Gene Technology Co., Ltd), and subjected to 1.5% agarose gel electrophoresis. With DL2000 Plus DNAMarker from Vazyme as a control, a band of specific PCR product was observed in a gel imaging system. The band of the PCR product was clear and specific. The size of the fragment was in agreement with the size of the product ZYT-CD36 cDNA fragment amplified in the step 1. 5 L of the PCR product was sequenced. The cDNA sequencing results of the 220T-CD36-CHO-K1 line show that the sequence is in agreement with the sequence of the CD36 gene fragment in the transfected plasmid 220T-CD36-EGFP-pLV4/StripII-HIS10, as shown in FIG. 8. This suggests that the established 220T-CD36-CHO-K1 line can accurately express the mutant CD36 220C>T gene at a molecular level. The cDNA sequencing results of the positive control Normal-CD36-CHO-K1 line show that the sequence is in agreement with the sequence of the CD36 gene fragment in the transfected plasmid Normal-CD36-EGFP-pLV4/StripII-HIS10. This suggests that the established Normal-CD36-CHO-K1 line can accurately express normal CD36 gene at molecular level. The cDNA sequencing results of the negative control EGFP-pLV4-CHO-K1 line show that the sequence is in agreement with the sequence of the EGFP gene fragment in the transfected plasmid EGFP-pLV4/StripII-HIS 10. This suggests that the established EGFP-pLV4-CHO-K1 line can accurately express the EGFP gene at molecular level.

[0117] The 220T-CD36-CHO-K1 line, the positive control Normal-CD36-CHO-K1 line, the negative control EGFP-pLV4-CHO-K1 line, and the blank control CHO-K1 line in logarithmic growth phase were collected, and detected for the CD36 expression by flow cytometry. The detection results are shown in FIG. 9, and indicate that the 220T-CD36-CHO-K1 line, the negative control EGFP-pLV4-CHO-K1 line, and the blank control CHO-K1 line are all negative in CD36 expression, and the positive control NORMAL-CD36-CHO-K1 line is positive in CD36 expression. Total membrance proteins were extracted from the several cell lines, and the expression of the CD36 protein by each cell line was detected by western-blotting. The detection results are shown in FIG. 10 and indicate that the 220T-CD36-CHO-K1 line, the negative control EGFP-pLV4-CHO-K1 line, and the blank control CHO-K1 line are negative in CD36 expression, and the positive control Normal-CD36-CHO-K1 line is positive in CD36 expression.

[0118] Unless otherwise indicated, the numerical ranges involved in the invention include the end values. While particular embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects, and therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention.