Eukaryotic expression and bioactivity verification of porcine prolactin
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摘要:目的
催乳素(Prolactin，PRL)具有广泛的生理调节作用，但其多效性机制仍不清楚。为了更好地研究猪PRL的多效性，本研究制备猪源PRL真核重组蛋白并验证其生物活性。
方法利用分子克隆技术将猪PRL基因克隆到慢病毒表达载体pCDH-CMV-MCS-EF1-GFP+Puro中，经慢病毒包装获得携带猪PRL基因的PRL−慢病毒；用浓缩的PRL−慢病毒感染CHO-K1细胞，经嘌呤霉素筛选后，获得能够分泌PRL重组蛋白的阳性细胞系CHO-K1-PRL；利用镍柱亲和层析法对重组蛋白进行纯化并进行LC-MS/MS质谱鉴定，利用HC11细胞体外培养体系验证PRL重组蛋白的生物活性。
结果成功构建了携带猪PRL基因的pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro慢病毒表达载体；包装及浓缩后的PRL−慢病毒滴度为9.9×108 TU/mL，其感染的CHO-K1细胞经嘌呤霉素筛选后得到阳性细胞系CHO-K1-PRL；从CHO-K1-PRL细胞培养液中成功纯化出重组蛋白，质量浓度为50 μg/mL，LC-MS/MS质谱分析的覆盖率达94%，鉴定为猪PRL重组蛋白；重组PRL具有促进HC11细胞增殖及酪蛋白表达的生物活性。
结论构建的细胞系CHO-K1-PRL可稳定表达具有生物活性的猪重组PRL，为猪PRL功能的研究和生产应用奠定了基础。
Abstract:ObjectiveProlactin (PRL) has a wide range of physiological regulatory effects, but its pleiotropic mechanism is still unclear. In order to investigate the pleiotropy of porcine PRL, we obtain porcine PRL eukaryotic recombinant protein and verify its biological activity.
MethodThe porcine PRL gene was cloned into the lentiviral vector pCDH-CMV-MCS-EF1-GFP+Puro by molecular cloning technology, and the PRL-lentivirus carrying porcine PRL gene was obtained by lentivirus packaging. CHO-K1 cells were infected by the concentrated PRL-lentivirus solution, and the positive cell line named CHO-K1-PRL, which could secrete recombinant PRL protein, was obtained after purinomycin screening. The recombinant protein was purified by nickel column affinity chromatography, and identified by LC-MS/MS mass spectrometry. The biological activity of recombinant PRL was verified by adding recombinant PRL into HC11 cell culture system in vitro.
ResultThe recombinant expression vector pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro carrying porcine PRL gene was successfully constructed. The titer of PRL-lentivirus after concentrating was 9.9×108 TU/mL, and the positive cell line CHO-K1-PRL was obtained after puromycin screening. The recombinant protein with the mass concentration of 50 μg/mL was successfully purified from the supernatant of CHO-K1-PRL cells. The recombinant porcine PRL protein was identified as porcine PRL by LC-MS/MS mass spectrometry, and the coverage rate of recombinant PRL was 94%. The recombinant PRL had the biological activity of promoting the proliferation and casein expression of HC11 cells.
ConclusionThe cell line CHO-K1-PRL constructed in this study can stably express porcine recombinant PRL with biological activity, which lays a foundation for the functional research, production and application of porcine PRL.
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Keywords:
- Porcine /
- Prolactin /
- CHO-K1 cell /
- Eukaryotic expression /
- Lentiviral vector /
- Recombinant protein
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图 1 pMD18-T-PRL克隆载体的构建与PCR鉴定
A：PRL基因的PCR产物，M为DL 2000 DNA Marker，1~4以母猪垂体组织cDNA为模板；B：pMD18-T-PRL菌液PCR产物，M为DL 2000 DNA Marker，1~16分别以挑选的不同pMD18-T-PRL菌落的菌液为模板
Figure 1. Construction and PCR identification of pMD18-T-PRL cloning plasmid
A: PCR products of PRL gene, M is DL 2000 DNA Marker, 1−4 use sow pituitary tissue cDNA as template; B: PCR products of pMD18-T-PRL colonies, M is DL 2000 DNA Marker, 1−16 respectively use different pMD18-T-PRL colonies as template
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图 2 pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro慢病毒表达载体的构建与PCR鉴定
A：EcoR I-6His-PRL-6His-BamH I序列片段PCR产物，M为DL 2000 DNA Marker，1为EcoR I-6His-PRL-6His-BamH I序列片段；B：EcoR I-6His-PRL-6His-BamH I序列片段双酶切产物，M为DL 2000 DNA Marker，1为6His-PRL-6His片段；C：pCDH-CMV-MCS-EF1-GFP+Puro质粒双酶切产物，M为DL 15000 DNA Marker，1为pCDH-CMV-MCS-EF1-GFP+Puro线性片段；D：pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro菌液PCR产物，M为DL 2000 DNA Marker，1~12分别以挑选的不同单菌落的菌液为模板；E：重组质粒pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro的双酶切结果，M1为DL 15000 DNA Marker，M2为DL 2000 DNA Marker，1和2为重组质粒双酶切片段
Figure 2. Construction and PCR identification of pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro lentivirus expression plasmid
A: PCR product of EcoR I-6His-PRL-6His-BamH I sequence fragment, M is DL 2000 DNA Marker, 1 is EcoR I-6His-PRL-6His-BamH I sequence fragment; B: Double digested product of EcoR I-6His-PRL-6His-BamH I sequence fragment, M is DL 2000 DNA Marker, 1 is 6His-PRL-6His sequence fragment; C: Double digested product of pCDH-CMV-MCS-EF1-GFP+Puro plasmid, M is DL 15000 DNA Marker, 1 is pCDH-CMV-MCS-EF1-GFP+Puro plasmid linear fragment; D: PCR products of pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro colonies, M is DL 2000 DNA Marker, 1−12 respectively use selected bacterial solutions from different single colonies as templates; E: Double digested product of recombinant vector pCDH-CMV-MCS-EF1-GFP+Puro, M1 is DL 15000 DNA Marker, M2 is DL 2000 DNA Marker, 1 and 2 are double digested products of recombinant vector
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图 3 慢病毒载体转染293T细胞24 h荧光表达情况
空白：未做处理的293T细胞；空载：转染空慢病毒载体的293T细胞；PRL：转染pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro慢病毒表达载体的293T细胞
Figure 3. Fluorescent expression of 293T cells transfected with lentivirus plasmid for 24 h
Control: Untreated 293T cells; Empty vector: 293T cells transfected with empty lentiviral vector; PRL: 293T cells transfected with pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro lentiviral vector
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图 4 慢病毒浓缩液感染CHO-K1细胞72 h及药筛7 d后荧光表达情况
空白：未做处理的CHO-K1细胞；空载：感染空载体慢病毒液的CHO-K1细胞；PRL：感染PRL−慢病毒液的CHO-K1细胞；PRL(药筛后)：感染PRL−慢病毒液的CHO-K1细胞经嘌呤霉素药物筛选7 d后
Figure 4. Fluorescence expression of CHO-K1 cells infected with concentrated lentivirus solution for 72 h and seven days after drug screening
Control: Untreated CHO-K1 cells; Empty vector: CHO-K1 cells infected by the concentrated empty lentivirus solution; PRL: CHO-K1 cells infected by the concentrated PRL-lentivirus solution; PRL (after drug screening): CHO-K1 cells infected with PRL-lentivirus solution and then screened by purinomycin for seven days
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图 5 CHO-K1-PRL细胞的RT-PCR鉴定
M为DL 2000 DNA Marker； 1、3、4、6和7为749 bp 的EcoR I-6His-PRL-6His-BamH I片段，2、5和8为98 bp的β-actin
Figure 5. RT-PCR identification of CHO-K1-PRL cells
M is DL 2000 DNA Marker; 1, 3, 4, 6 and 7 are the 749 bp EcoR I-6His-PRL-6His-BamH I fragments; 2, 5 and 8 are the 98 bp β-actin fragments
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图 6 CHO-K1-PRL细胞中His-Tag蛋白Western blot鉴定
A：细胞裂解液：B：细胞培养液；M为Western blot marker，1为未做处理的CHO-K1细胞，2为感染空载体慢病毒液的CHO-K1细胞，3为CHO-K1-PRL；PRL-6His重组蛋白和内参α-tubulin相对分子质量分别约为25 000和55 000
Figure 6. Western blot identification of His-Tag protein in CHO-K1-PRL cells
A: Cell lysate; B: Cell culture fluid; M is Western blot marker, 1 is untreated CHO-K1 cells, 2 is CHO-K1 cells infected by the concentrated empty lentivirus solution, 3 is CHO-K1-PRL; The relative molecular weights of PRL-6His recombinant protein and α-tubulin are about 25 000 and 55 000 resprectively
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图 7 纯化猪源PRL重组蛋白的SDS-PAGE鉴定
Figure 7. SDS-PAGE identification of purified porcine PRL recombinant proteins
M: Protein marker (PM2510)
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图 8 不同质量浓度重组PRL对HC11细胞增殖及酪蛋白表达的影响
柱子上方(图A)、相同蛋白(图B)或基因(图C)柱子上方的不同小写字母表示组间差异显著(P<0.05，LSD法)，不同大写字母表示组间差异极显著(P<0.01，LSD法)
Figure 8. Different mass concentrations of recombinant PRL on proliferation and casein expressions of HC11 cells
Different lowercase letters on bars (figure A), or bars of the same protein (figure B) or gene (figure C) indicate significant differences among groups (P<0.05, LSD method), different capital letters indicate highly significant differences among groups (P<0.01, LSD method)
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图 9 Western blot检测不同质量浓度重组PRL对HC11细胞中Cyclin D1、CSN2蛋白表达的影响
Figure 9. Effects of different mass concentrations of recombinant PRL on expressions of Cyclin D1 and CSN2 proteins in HC11 cells detected by western blot
表 1 qPCR引物序列
Table 1 Primer sequence for qPCR
基因
Gene正向引物序列(5′→ 3′)
Forward primer sequence反向引物序列(5′→ 3′)
Reverse primer sequence产物大小/bp
Product lengthGAPDH GAGCGAGACCCCACTAACATC GCGGAGATGATGACCCTTTT 134 PRLR GTGGAATCCTGGGTCAGATG GGGCCACTGGTTTTGTAGTC 108 CSN1S1 CCTTTCCCCTTTGGGCTTAC TGAGGTGGATGGAGAATGGA 193 CSN2 GCAATCCCGTCCCACAAAAC GGGGCATCTGTTTGTGCTTG 138 CSN3 CCTTTTTGTGCCGTGGTGAG GGCTGGAGACCTAAGCAGAA 197 Cyclin D1 TGGCTAAACAAGGACCCCC ATGTCCACATCTCGCACGTC 203 PCNA AAAGATGCCGTCGGGTGAAT TGGTTACCGCCTCCTCTTCT 179 
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