Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (12): 801-808.DOI: 10.12016/j.issn.2096-1456.2021.12.002
• Basic Study • Previous Articles Next Articles
LAI Yangfan1(),WANG Peng1,QIAO Li2,LIU Zhongjing2,YE Zhaoyang2(
),LIANG Yan1(
)
Received:
2021-02-02
Revised:
2021-04-23
Online:
2021-12-20
Published:
2021-08-17
Contact:
Zhaoyang YE,Yan LIANG
Supported by:
赖扬帆1(),王鹏1,乔里2,刘中静2,叶朝阳2(
),梁燕1(
)
通讯作者:
叶朝阳,梁燕
作者简介:
赖扬帆,医师,硕士研究生,Email: 基金资助:
CLC Number:
LAI Yangfan,WANG Peng,QIAO Li,LIU Zhongjing,YE Zhaoyang,LIANG Yan. Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(12): 801-808.
赖扬帆,王鹏,乔里,刘中静,叶朝阳,梁燕. 变异链球菌hit基因缺陷菌株的构建[J]. 口腔疾病防治, 2021, 29(12): 801-808.
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Primers | Sequence (5′-3′) |
---|---|
hit(Up) | F:CCGCTCGAGTCAACCCTCTTTAGTCAAAGTCATAT(XhoI) |
R:CCCAAGCTTGTCCTCATAAACTTTTGATAGAGGGAATGT(HindIII) | |
hit(Down) | F:GGAATTCCATATGCCTGACTTTACCCGTCTTGGACAAT(NdeI) |
R:GGACTAGTAAATAAAAAATAAGGTGGAAGGGTAT(SpeI) | |
pFW5(PCR) | F:AGGATGAGGAGGCAGATTGCCTTGAATAT |
R1:ATTCCTCTGACGAATCCATAATGGCTCTT | |
F1:ATATACGGAAATTATGACTTAGAGGAATT | |
R:CGCAGCGAGAAAAAAGGCCCACTTTTGT | |
hit(PCR) | F:CATGACGTTTCTTTCTAACTTTGATAT |
R:GTTTGTAATACATCTGATAAAGACCGGCT |
Table 1 Primers and sequences used in the experiment
Primers | Sequence (5′-3′) |
---|---|
hit(Up) | F:CCGCTCGAGTCAACCCTCTTTAGTCAAAGTCATAT(XhoI) |
R:CCCAAGCTTGTCCTCATAAACTTTTGATAGAGGGAATGT(HindIII) | |
hit(Down) | F:GGAATTCCATATGCCTGACTTTACCCGTCTTGGACAAT(NdeI) |
R:GGACTAGTAAATAAAAAATAAGGTGGAAGGGTAT(SpeI) | |
pFW5(PCR) | F:AGGATGAGGAGGCAGATTGCCTTGAATAT |
R1:ATTCCTCTGACGAATCCATAATGGCTCTT | |
F1:ATATACGGAAATTATGACTTAGAGGAATT | |
R:CGCAGCGAGAAAAAAGGCCCACTTTTGT | |
hit(PCR) | F:CATGACGTTTCTTTCTAACTTTGATAT |
R:GTTTGTAATACATCTGATAAAGACCGGCT |
Figure 1 Electrophoresis of PCR products of the upstream and downstream sequences of the hit gene and the circle map of the pFW5 vector a: DNA electrophoresis of PCR products of the upstream and downstream DNA fragments of the hit gene; M: marker; Up: the upstream DNA fragment of approximately 850 bp; Down: the downstream DNA fragment of approximately 500 bp; b: the vector pFW5 carries two multiple cloning sites, MCS-I and MCS-II, which are separated by the aad9 spectinomycin-resistance gene sequence
Figure 2 Electrophoresis of PCR- and double digested-products of the recombinant plasmid pFW5 a: DNA electrophoresis of the PCR products of the MCS-Ⅰ- and MCS-Ⅱ- region DNA fragments of the recombinant plasmids pFW5 (20# and 141#); b: DNA electrophoresis of the products (20_x/s and 141_x/s) of the recombinant plasmids pFW5 (20# and 141#) digested by XhoI and SpeI. The pFW5 vector and pFW5_x/s served as the control. M: Marker
Figure 3 DNA electrophoresis of PCR products of the hit gene DNA fragments from the Streptococcus mutans genome of plasmid (20#) genetic transformation Lanes 20-3, 20-4 to 20-24 correspond to 19 random positive colony forming units on an aad9 spectinomycin-resistant BHI agar plate, respectively
Figure 4 Electrophoresis and Sanger sequencing of PCR products of the hit gene fragment from the hit-deficient mutant strains and their parental S. mutans ATCC25175 a: DNA electrophoresis of PCR products of the hit gene DNA fragments of the 20-3# and 20-5# strains; b: Sanger forward sequencing chromatograms of the PCR products of 20-3# strain (the top panel) and its parent strain ATCC25175 (the down panel); c: DNA electrophoresis of the PCR products of the hit gene DNA fragments of Streptococcus mutans ATCC25175; d: Sanger reversed sequencing chromatograms of the PCR products of 20-3# strain (the top panel) and its parent strain ATCC25175 (the down panel)
Figure 5 Electrophoresis and Sanger sequencing chromatograms of the PCR products of the hit gene DNA fragments from the hit-deficient mutant strains a: DNA electrophoresis of PCR products of the hit gene DNA fragments of the 1-1-1-15 and 6-1 strains. b&c: sanger forward sequencing (b) and reversed sequencing chromatograms (c) of the PCR products of 6-1 strains
Figure 6 Growth curve and growth rate of the hit-deficient mutant strains versus Streptococcus mutans a: the hit-deficient mutant strains (Smu.Δhit) and their parent strains ATCC25175 have similar growth curves; b: the growth rate of the hit-deficient mutant strains (Smu.Δhit) is much faster than that of their parent strains ATCC25175 at 48-hour intervals
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