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CN111909964A - Method for efficiently expressing AFP3-CASP3 fusion protein - Google Patents

Method for efficiently expressing AFP3-CASP3 fusion protein Download PDF

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CN111909964A
CN111909964A CN202010862809.0A CN202010862809A CN111909964A CN 111909964 A CN111909964 A CN 111909964A CN 202010862809 A CN202010862809 A CN 202010862809A CN 111909964 A CN111909964 A CN 111909964A
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casp3
afp3
fusion protein
hbx
lentivirus
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刘坤
林波
朱明月
李伟
董栩
李孟森
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Hainan Medical College
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Hainan Medical College
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Abstract

The invention discloses a method for efficiently expressing AFP3-CASP3 fusion protein, belonging to the technical field of biology, the invention clones HBx gene into lentivirus, human hepatoma cells are infected by the lentivirus-HBx, after the HBx protein is expressed in the hepatoma cells, an expression vector containing AFP3 rd structural domain and CASP3 gene is transfected into the human hepatoma cells expressing the HBx protein, the HBx protein is expressed in the hepatoma cells, the HBx protein promotes the efficient secretory expression of the human AFP3-CASP3 fusion protein, and the AFP3-CASP3 fusion protein is obtained by concentrating and purifying culture medium. The AFP3-CASP3 fusion protein expressed by HLE, HepG2 or Bel-7402 liver cancer cells promotes apoptosis, the apoptosis capacity is increased by 45-52%, and when the AFP treats the liver cancer cells, the apoptosis capacity is obviously reduced by 22-26%. The AFP3-CASP3 fusion protein obtained by the invention can be used for research of tumor treatment.

Description

Method for efficiently expressing AFP3-CASP3 fusion protein
Technical Field
The invention belongs to the technical field of biology, relates to genetic engineering and protein engineering, and particularly relates to a method for efficiently carrying out fusion expression and purification on a third structural domain of AFP (human alpha-fetoprotein) and CASP3 (cysteine protease 3).
Background
Alpha-fetoprotein (AFP) gene is openly expressed during fetal development, but is basically in a closed state two years after human birth, but when adult develops liver cancer, the AFP gene is reactivated and abundantly expressed, so that AFP is used as a standard for diagnosing liver cancer, and AFP can enter cancer cells through its receptor.
The most important terminal cutter in the apoptosis of CASP3 cells is also an important component of the CTL cell killing mechanism. Expression of the AFP3-CASP3 fusion protein helps to bring CASP3 protein into cancer cells through the 3 rd domain of AFP, thereby promoting apoptosis of cancer cells.
At present, no report about a method for efficiently expressing and purifying a fusion protein AFP3-CASP3 by using liver cancer cells is found through retrieval.
Disclosure of Invention
The invention aims to provide a method for efficiently expressing and purifying AFP3-CASP3 fusion protein by using liver cancer cells, the expressed protein has better activity and can be used for researching medicaments for promoting the apoptosis of the liver cancer cells.
In order to achieve the purpose, the technical scheme of the invention is as follows: a method for efficiently expressing an AFP3-CASP3 fusion protein is provided, wherein: the HBx gene is cloned into lentivirus, a human liver cancer cell is infected by the lentivirus-HBx, after HBx protein is expressed in the liver cancer cell, an expression vector containing the 3 rd domain of AFP and CASP3 gene is transfected into the human liver cancer cell expressing the HBx protein, the HBx protein is expressed in the liver cancer cell, the HBx protein promotes the efficient secretory expression of the human AFP3-CASP3 fusion protein, and the AFP3-CASP3 fusion protein is obtained by concentrating and purifying a culture medium.
Further, the method for efficiently expressing the AFP3-CASP3 fusion protein comprises the following steps:
(1) construction of expression vectors
a. Cloning AFP3 rd structural domain gene (genes of 401-610 residues, specifically synthesized gene sequences are shown as SEQ ID NO:1 and SEQ ID NO:2 in a sequence table) and full-length CASP3 gene (specifically synthesized gene sequences are shown as SEQ ID NO:3 and SEQ ID NO:4 in the sequence table) into an expression vector PTT5 vector, adding 6 histidine sequences to the C end of the expression vector, converting DH5 alpha cells, and extracting plasmids to obtain PTT5-AFP3-CASP3 plasmids;
b. cloning an HBx full-length gene (NCBI accession number is AB210819) into a lentivirus expression vector, transfecting 293T cells by utilizing a three-plasmid system pHBLV-HBx, pSPAX2 and pMD2G, respectively collecting virus supernatants of 48h and 72h, and ultracentrifuging to obtain a lentivirus concentrated solution, namely lentivirus-HBx;
(2) transfection of expression vectors into human hepatoma cells
Infecting human hepatoma cells with lentivirus-HBx for 72 hours, screening out a stable expression strain by puromycin, transfecting PTT5-AFP3-CASP3 plasmid, and collecting a culture medium after 48-96 hours;
(3) purification of AFP3-CASP3 fusion protein
Adding HBS (10mM Hepes, pH7.2, 150mM NaCl) solution into the above collected culture medium, concentrating through a filter membrane, adsorbing the concentrated solution onto a nickel column, eluting with an eluent, purifying the eluent through a gel column, further concentrating through a filter membrane, and freeze-drying to obtain the AFP3-CASP3 fusion protein.
Further, the molecular weight of the AFP3-CASP3 fusion protein is 55-60 KD; compared with human alpha-fetoprotein (AFP), AFP inhibits apoptosis, while AFP3-CASP3 fusion protein promotes apoptosis.
Further, the liver cancer cell line is HLE, HepG2 or Bel-7402 liver cancer cell which is used for experiments and has no infection related report; the lentivirus is used for experiments and has no infection related reports.
Further, the puromycin concentration is 1.0. mu.g/mL.
Furthermore, the liver cancer cell is a HBx-transfected lentivirus expression vector and stably expresses HBx protein.
The obtained AFP3-CASP3 fusion protein is provided with CASP3 protein behind the 3 rd structural domain of AFP, so that CASP3 protein is brought into cancer cells through the 3 rd structural domain of AFP, thereby promoting the apoptosis of the cancer cells.
The AFP3-CASP3 fusion protein expressed by HLE, HepG2 or Bel-7402 liver cancer cells promotes apoptosis, the apoptosis capacity is increased by 45-52%, and when the AFP treats the liver cancer cells, the apoptosis capacity is obviously reduced by 22-26%.
The AFP3-CASP3 fusion protein obtained by the invention can be used for research of tumor treatment.
Drawings
FIG. 1: flow cytometry is used for observing that AFP3-CASP3 fusion protein can promote apoptosis of liver cancer cells. According to flow cytometry data analysis, the apoptosis rates of early stage and late stage of Bel-7402, HLE and HepG2 are respectively as follows: 20%, 22%, 23%. After 20 mu g/mL AFP is added for 48 hours, the apoptosis rates are respectively 15%, 16.2% and 18%. After 20 mu g/mL AFP3-CASP3 fusion protein is added to act for 48 hours, the apoptosis rates are respectively 30%, 32% and 35%. The AFP can be used for treating the liver cancer cells, and the apoptosis capacity of the cells is obviously reduced by 22-26%. The AFP3-CASP3 fusion protein expressed by HLE, HepG2 or Bel-7402 liver cancer cells promotes apoptosis, and the apoptosis capacity is increased by 45-52%.
Detailed Description
Example A method for high-efficiency expression and purification of AFP3-CASP3 fusion protein by using HLE cells
1. Establishment of HBx-expressing HLE cells
According to a human HBx gene sequence (NCBI accession number is AB210819), HBx is constructed into a lentivirus expression vector pHBLV through enzyme digestion and ligation reaction, a three-plasmid system pHBLV-HBx, pSPAX2 and pMD2G are used for transfecting 293T cells, virus supernatants of 48h and 72h are respectively collected, an ultracentrifugation is carried out to obtain a lentivirus concentrated solution (called lentivirus-HBx), then HLE cells are infected, and after 72 hours of culture, the lentivirus concentrated solution is screened by 1.0 mu g/mL puromycin to obtain a stable expression cell strain.
2. The plasmid containing PTT5-AFP3-CASP3 is transfected into HLE cells expressing HBx, and after 48 hours of culture, the culture medium is collected.
3. AFP3-CASP3 fusion protein is separated, purified and identified.
Since the protein is expressed secretly, the supernatant was collected by centrifugation at 8000r/min for 5min, and the solution was concentrated by cutting to a flow membrane, and the solution was HBS buffer (10mM Hepes, pH7.2, 150mM NaCl). Then, a nickel column was hung and washed with 300mmol/L imidazole. Purification by gel chromatography (AKAT, spudex200 column) was then continued with HBS (10mM Hepes, pH7.2, 150mM NaCl) as mobile phase. The molecular weight of the expressed fusion protein is about 55-60 KD.
4. HLE-expressed AFP3-CASP3 fusion proteins can inhibit cancer cell growth.
The AFP-CASP3 fusion protein is observed to promote the apoptosis of liver cancer cells through flow cytometry: according to flow cytometry data analysis, the early plus late apoptosis rate of HLE was: 22 percent. After 20 mu g/mL AFP is added and acted for 48 hours, the apoptosis rate is 16.2 percent. After 20 mu g/mL HLE expression AFP3-CASP3 fusion protein is added for acting for 48 hours, the apoptosis rate is 32%. Therefore, when AFP is used for treating HLE liver cancer cells, the apoptosis capacity is obviously reduced by about 26 percent. The AFP3-CASP3 fusion protein expressed by the HLE of the liver cancer cell promotes apoptosis, and the apoptosis capacity is increased by 45 percent (shown in figure 1).
Example two method for efficiently expressing and purifying AFP3-CASP3 fusion protein by using HepG2 cell
1. Establishment of HBx-expressing HepG2 cells
According to a human HBx gene sequence (NCBI accession number is AB210819), HBx is constructed into a lentivirus expression vector pHBLV through enzyme digestion and ligation reaction, a three-plasmid system pHBLV-HBx, pSPAX2 and pMD2G are used for transfecting 293T cells, virus supernatants of 48h and 72h are respectively collected, an ultracentrifugation is carried out to obtain a lentivirus concentrated solution (called lentivirus-HBx), HepG2 cells are infected, and the lentivirus concentrated solution is screened by 1.0 mu g/mL puromycin after being cultured for 72 hours to obtain a stable expression cell strain.
2. The plasmid containing PTT5-AFP3-CASP3 is transfected into HepG2 cells expressing HBx, and after 96 hours of culture, the culture medium is collected.
3. AFP3-CASP3 fusion protein is separated, purified and identified. Since the protein is expressed secretly, the supernatant was collected by cutting the flow-through membrane after centrifuging the medium and cell mixture at 8000r/min for 5min, and the solution was HBS buffer (10mM Hepes, pH7.2, 150mM NaCl). Then, a nickel column was hung and washed with 300mmol/L imidazole. Purification by gel chromatography (AKAT, spudex200 column) was then continued with HBS (10mM Hepes, pH7.2, 150mM NaCl) as mobile phase. The molecular weight of the expressed fusion protein is about 55-60 KD.
4. The AFP3-CASP3 fusion protein expressed by HepG2 can inhibit the growth of cancer cells.
It is observed by flow cytometry that AFP3-CASP3 fusion protein can promote apoptosis of hepatoma cells: according to flow cytometry data analysis, the apoptosis rate of HepG2 at early and late stages was: 23 percent. After 20 mu g/mL AFP is added and acted for 48 hours, the apoptosis rate is 18 percent. After 20 mu g/mL of AFP3-CASP3 fusion protein expressed by HepG2 is added for acting for 48 hours, the apoptosis rate is 35 percent. It can be seen that AFP can obviously reduce about 22% of apoptosis ability when treating liver cancer cells. The AFP-2CASP3 fusion protein expressed by HepG2 liver cancer cells promotes apoptosis, and the apoptosis capacity is increased by about 52 percent (shown in figure 1).
EXAMPLE III method for efficient expression and purification of AFP3-CASP3 fusion protein Using Bel-7402 cells
1. Establishment of HBx-expressing Bel-7402 cells
According to a human HBx gene sequence (NCBI accession number is AB210819), HBx is constructed into a lentivirus expression vector pHBLV through enzyme digestion and ligation reaction, a three-plasmid system pHBLV-HBx, pSPAX2 and pMD2G are used for transfecting 293T cells, virus supernatants of 48h and 72h are respectively collected, an ultracentrifugation is carried out to obtain a lentivirus concentrated solution (called lentivirus-HBx), then Bel-7402 cells are infected, and after 72 hours of culture, puromycin of 1.0 mu g/mL is used for screening to obtain a stable expression cell strain.
2. The plasmid containing PTT5-AFP3-CASP3 is transfected into Bel-7402 cells expressing HBx, and after 85 hours of culture, the culture medium is collected.
3. AFP3-CASP3 fusion protein is separated, purified and identified. Since the protein is expressed secretly, the supernatant was collected by cutting the flow-through membrane after centrifuging the medium and cell mixture at 8000r/min for 5min, and the solution was HBS buffer (10mM Hepes, pH7.2, 150mM NaCl). Then, a nickel column was hung and washed with 300mmol/L imidazole. Purification by gel chromatography (AKAT, spudex200 column) was then continued with HBS (10mM Hepes, pH7.2, 150mM NaCl) as mobile phase. The molecular weight of the expressed fusion protein is about 55-60 KD.
4. The AFP3-CASP3 fusion protein expressed by Bel-7402 can inhibit the growth of cancer cells.
It is observed by flow cytometry that AFP3-CASP3 fusion protein can promote apoptosis of hepatoma cells: according to flow cytometry data analysis, the apoptosis rate of early and late Bel-7402 is as follows: 20 percent. After 20 mu g/mL AFP is added and acted for 48 hours, the apoptosis rate is 15 percent. After 20 mu g/mL HLE expression AFP3-CASP3 fusion protein is added for acting for 48 hours, the apoptosis rate is 30 percent. It can be seen that AFP treatment of liver cancer cell can reduce apoptosis obviously by 25%. The AFP3-CASP3 fusion protein expressed by Bel-7402 liver cancer cells promotes apoptosis, and the apoptosis capacity is increased by 50 percent (figure 1).
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.
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Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe
245 250 255
His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu
260 265 270
Leu Tyr Phe Tyr His His His His His His His
275 280

Claims (5)

1. A method for efficiently expressing AFP3-CASP3 fusion protein is characterized in that: the HBx gene is cloned into constructed lentivirus, a human liver cancer cell is infected by the lentivirus-HBx, HBx protein is expressed in the liver cancer cell, an expression vector containing the 3 rd domain of AFP and CASP3 gene is transfected into the human liver cancer cell expressing the HBx protein, the HBx protein is expressed in the liver cancer cell, the HBx protein promotes the high-efficiency secretion expression of the human AFP3-CASP3 fusion protein, and the AFP3-CASP3 fusion protein is obtained by concentrating and purifying a culture medium.
2. The method for efficiently expressing AFP3-CASP3 fusion protein according to claim 1, which comprises the following steps:
(1) construction of expression vectors
a. Cloning AFP3 rd structural domain gene and full-length CASP3 gene into expression vector PTT5, adding 6 histidine sequences at C end, transforming DH5 alpha cells, and extracting plasmid to obtain PTT5-AFP3-CASP3 plasmid;
b. cloning the HBx full-length gene into a lentivirus expression vector, transfecting 293T cells by utilizing three plasmid systems pHBLV-HBx, pSPAX2 and pMD2G, respectively collecting virus supernatants of 48h and 72h, and ultracentrifuging to obtain a lentivirus concentrated solution, namely lentivirus-HBx;
(2) transfection of expression vectors into human hepatoma cells
Infecting human hepatoma cells with lentivirus-HBx for 72 hours, screening out a stable expression strain by puromycin, transfecting PTT5-AFP3-CASP3 plasmid, and collecting a culture medium after 48-96 hours;
(3) purification of AFP3-CASP3 fusion protein
Adding the collected culture medium into HBS solution, concentrating with filter membrane, adsorbing the concentrated solution onto nickel column, eluting with eluent, purifying the eluent with gel column, further concentrating with filter membrane, and freeze drying to obtain AFP3-CASP3 fusion protein.
3. The method for efficiently expressing the AFP3-CASP3 fusion protein as claimed in claim 1 or 2, wherein: the AFP3-CASP3 fusion protein has the molecular weight of 55-60 KD.
4. The method for efficiently expressing the AFP3-CASP3 fusion protein as claimed in claim 1 or 2, wherein: the liver cancer cell line is HLE, HepG2 or Bel-7402 liver cancer cell which is used for experiments and has no infection related report; the lentivirus is used for experiments and has no infection related reports.
5. The method for efficiently expressing an AFP3-CASP3 fusion protein as claimed in claim 2, wherein: the puromycin concentration is 1.0 mug/mL.
CN202010862809.0A 2020-08-25 2020-08-25 Method for efficiently expressing AFP3-CASP3 fusion protein Pending CN111909964A (en)

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Application publication date: 20201110