Phagemid vectorwith high-density and application thereof
A phagemid and carrier technology, applied in the biological field, can solve the problems of reducing the theoretical library capacity of the polypeptide library, affecting the acquisition and loss of functional polypeptides, etc., and achieve the effects of reducing genetic preference, high electrotransformation efficiency, and improving success rate.
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Embodiment 1
[0030] Construction pComb-pVIII phagemid: First Example
[0031] Oligonucleotides were annealed by the method of extension using primers SEQ ID NO 1 and SEQ ID NO 2 M13 phage pVIII synthetic signal peptide gene-containing restriction sites Kpn I (fragment 1), using primers SEQ ID NO 3 and SEQ ID NO4 synthesis containing Xho I restriction sites of the M13 phage pVIII protein gene (fragment 2), sequentially inserted pComb3xss phagemid (see gene map figure 1 -A), the pComb-pVIII phagemid construct (see Gene Map figure 1 -B). Primer sequences are shown in Table 1.
[0032] Table 1 pComb-pVIII phagemid construct using primers
[0033]
[0034]
[0035] 1, electroporation
[0036] (1) ER2738 prepared electrocompetent removed from the freezer -80 ℃, -20 ℃ extracted out of the refrigerator prechilled 0.1cm cuvette, inserted immediately on ice, so that waiting for 5min competent thawed electroporation apparatus is set in advance for the condition 1.8 KV, 4ms;
[0037] (2) in the clean ...
Embodiment 2
[0065] Example 2: Construction of phage display random cyclic 8, 9, 10 peptide library
[0066] The random 8, 9, 10 peptide gene encoded by the NNK is synthesized by the primer SEQ ID NO 6 and SEQ ID NO 7 to 9, respectively, and PCOMB is inserted by XHO I and KPN I. -PVIII Phage granules, the random polypeptide sequence is located between the PVIII protein N end of the phage PVIII protein and its signal peptide. The primers used are shown in Table 2, and the specific steps are as follows:
[0067] Table 2 Phage show the primer used in randomized annular 8, 9, 10 peptide library
[0068]
[0069] 1. Synthesis, insertion of random annular 8, 9, 10 peptide gene
[0070] (1) annealing
[0071]
[0072] The reaction system was 50 μl, 95 ° C for 20 min, and slowly annealed to room temperature.
[0073] (2) Extension: System, steps are the same as in Examples 2 (2)
[0074] (3) Enzyme cutting of random cyclic 8, 9, 10 peptide genes: The restriction endonuclease used is KPN I and XHO ...
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