Expression and separation purification for S-protein of recombinant SARS coronavirus
A coronavirus, separation and purification technology, applied in the field of biology, can solve problems such as low expression level and no major breakthrough of S protein
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Embodiment 1
[0080] Amplification of SARS virus S protein gene
[0081] In the present embodiment, the S protein gene of SARS virus is amplified from the supernatant of Vero E6 cells infected with SARS BJ01 strain (available from the Academy of Military Sciences) by RT-PCR method, and cloned into pBluescriptSK (+) to obtain pBS ( See figure 1 ).
[0082] The specific process is as follows:
[0083] (a) The S gene is divided into three fragments and obtained by amplifying from the supernatant of SARS virus cell culture by the method of RT-PCR.
[0084] Fragment 1 (base sequence 1-1845) was amplified by the following primers
[0085] Upstream primer: 5'-GGGGAATTCAACATGTTTATTTTTCTTATTATTTCTTAC-3' (SEQ ID NO: 3)
[0086] Downstream primer: 5'-GGGGGTACCGAGTTGATCTGCATGAATTG-3' (SEQ ID NO: 4)
[0087] Fragment 2 (base sequence 1404-2646) was amplified by the following primers
[0088] Upstream primer: 5'-GGGGGAATTCCCCCACCTGCTCTTAATTGTTA-3' (SEQ ID NO: 5)
[0089] Downstream primer: 5'-GGGA...
Embodiment 2
[0100] Plasmid construction
[0101] In this example, according to the prediction results of the functional domains of the S protein, the appropriate restriction sites on pBS were used to directly or indirectly (transition with the pBluescriptKS(+) plasmid) clone into pGEX and pET32 plasmids.
[0102] Specifically, the Pst I-Sal I fragment S1b (751-1925bp) of the S protein gene was cloned into the corresponding restriction site of pBluescriptKS (+), and then the 2kb BamH I-Xho I fragment of the resulting clone was cloned into pET-32a got pS1b ( figure 1 );
[0103] EcoRI-Sal I fragment Sla (1-1925bp) is cloned into the corresponding enzyme cutting site of pGEX-4T1 to obtain pSla ( figure 1 );
[0104] The Sca I fragment S2 (2005-3410bp) of pBS was cloned into the Sma I restriction site of pGEX-3x to obtain pS2 ( figure 1 );
[0105] The Spe I fragment SΔ(32-3659bp) of pBS was connected with pBluescriptKS(+) to obtain the recombinant, and then its 3.7 Kb BamH I-NotI fragme...
Embodiment 3
[0109] expression condition
[0110] When detecting whether the target gene is expressed, when the OD value of the bacteria containing the expression vector is 1, induce it with 0.8mMIPTG, culture it at 28°C for 2 hours, collect the cells by centrifugation, suspend them in an appropriate amount of PBS buffer, and use Pressure cell press Press or sonicate cells. The initial cell extract was scanned and analyzed by SDS-PAGE and Gel Scan 4.0 software.
[0111] Unless otherwise specified, the medium used for expression was 2×YT. When a large amount of bacteria containing expression vectors pS1a, pS1b, pS2 and pSΔ are induced, when the OD value of the bacteria is 1, induce with 0.1mM IPTG, culture at 22°C for 4 hours, and the rest of the operations are the same as above.
[0112] When the cells containing the pS plasmid are cultured, the leaky expression product has certain cytotoxicity, so an additional 2% glucose is added to the culture medium. When the cells grow to an OD of 2...
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