Construction and preliminary application of the universal transfer vector in recombination of Bovine herpes virus type 1

doi:10.3969/j.issn.1004-1524.20221496

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (10): 2311-2320.DOI: 10.3969/j.issn.1004-1524.20221496

• Animal Science • Previous Articles Next Articles

Construction and preliminary application of the universal transfer vector in recombination of Bovine herpes virus type 1

DAI Shasha(), MA Xiaojing, WANG Jingsong, TIAN Xingmiao, WANG Jianlin, LI Jidong()

College of Animal Science, Ningxia University, Yinchuan 750021, China

Received:2022-10-26 Online:2023-10-25 Published:2023-10-31

Abstract

Abstract:

The study aimed to construct a universal transfer vector that could be used for recombination of Bovine herpesvirus type 1 (BHV-1). The vector prPP with bidirectional promoter and two multiple cloning sites was constructed by insertion of another CMV promoter at the reverse site of the pVAX1 CMV promoter. The universal transfer vector prPgP was constructed by putting the green fluorescent protein marker gene downstream the reverse CMV promoter. Using the BHV-1 glycoprotein gE as the recombination site, the gE upstream and downstream homology arm was inserted into two multiple cloning sites of the prPgP vector respectively. Following, the recombinant transfer vector p△gErPgP-E2 was constructed by placing the Bovine viral diarrhea virus(BVDV) E2 gene as exogenous gene under the forward CMV promoter. The plasmid p△gErPgP-E2 was transfected with Madin-Darby Bovine kidney (MDBK) infected with BHV-1 to generate recombinant viruses. The recombinant viruses were screened, purified, identified, and named as rBHV-1-△gE/E2. The report gene GFP and BVDV E2 was expressed normally by fluorescence microscopy. The results showed that the recombinant universal vector prPgP could be convenient for BHV-1 recombination and be used in related research and its vaccine development.

Key words: Bovine herpesvirus type 1, homologous arm, transfer vector, homologous recombination

CLC Number:

S855.3

DAI Shasha, MA Xiaojing, WANG Jingsong, TIAN Xingmiao, WANG Jianlin, LI Jidong. Construction and preliminary application of the universal transfer vector in recombination of Bovine herpes virus type 1[J]. Acta Agriculturae Zhejiangensis, 2023, 35(10): 2311-2320.

Figures/Tables 15

Fig.1 Schematic diagram of construction of universal vector prPgP prPP multiple cloning site (MCS) Ⅰ: SalⅠ, NsiⅠ, BglⅡ, EcoRⅠ, PstⅠ, EcoR V, NotⅠ, XhoⅠ, XbaⅠ, ApaⅠ; prPP multiple cloning site (MCS) Ⅱ: ClaⅠ, SaIⅠ, BspEⅠ, BamHⅠ, KpnⅠ, HindⅢ, AflⅡ, PmeⅠ, NheⅠ, XmaⅠ.

Table 1 Primer sequence and restriction site of prPgP vector

目的片段 Target fragment	引物序列 Primer sequence (5'→3')	酶切位点 Enzymatic cleavage site	退火温度 Annealing temperature/℃	长度 Length/bp
pVAX1-1	F: ATGCATAGATCTGAATTCTGCAGATATCCAG	NsiⅠ	55	2 966
	R: ATGCATCCGCGGCAGCTTGGGTCTCC	NsiⅠ
rCMV	F: AAAGTCGACTCCGGAGGATCCGAGCTCGGTAC	SalⅠ	60	629
	R: AAACCCGGGAATGGCCCGCCTGGCTG	XmaⅠ
pVAX1-2	F: TTTCCCGGGGACTCTTCGCGATGTAC	XmaⅠ	60	2 984
	R: AAAGTCGACATCGATAAGAACATGTGAGCAAAAG	SalⅠ
GFP	F: GGGCTTAAGTCACTTGTACAGCTCATCCA	AflⅡ	62	738
	R: AAAGCTAGCATGGTGAGCAAGGGCG	NheⅠ

Table 2 p△gErPgP-E2 plasmid primer sequence and restriction site

目的片段 Target fragment	引物序列 Primer sequence (5'→3')	酶切位点 Enzymatic cleavage site	退火温度/℃ Annealing temperature	长度 Length/bp
gE L	F: AAGGATCCCGCCGGGTTGTTAAATG	BamHⅠ	66	654
	R: GGAAGCTTGATGAGCCGGTCGTACA	HindⅢ
gE R	F: AACTGCAGCCACGTGGAAGCTTGGGA	PstⅠ	68	795
	R: TGATATCGCCTCATCAGCGCCTCGA	EcoRⅤ
E2	F: ATAAGATCTCGCCACCATGGTGCACTTGGATTGCAA	BglⅡ	60	1 156
	R: GCGCTGCAGCTACCCTAAGGCCTTCTGTTCTGATAA	PstⅠ

Fig.2 Schematic diagram of construction of recombinant vector p△gErPgP-E2

Table 3 Primer sequence for PCR identification of recombinant virus

目的片段 Target fragment	上游引物序列 Forward primer sequence (5'→3')	下游引物序列 Reverse primer sequence (5'→3')	退火温度 Annealing temperature/℃	长度 Length/bp
gE	ACTCAACGGGCGACAAAGAGTTT	TCTACGCTGTTATTGGCGGGAC	62	667
GFP	TTCACCTTGATGCCATTCTT	AGTGCTTCTCACGCTACCC	55	300
E2	CGCCACCATGGTGCACTTGGATTGCAA	CTACCCTAAGGCCTTCTGTTCTGATAA	60	1 144

Fig.3 Electrophoresis of PCR products of PRPGP vector target fragment M1, DL5000 DNA marker; 1, pVAX1-1; M2, DL2000 DNA marker; 2, rCMV; 3, pVAX1-2; M3, DL1000 DNA marker; 4, GFP gene.

Fig.4 Identification of recombinant plasmid prPP and prPgP by double restriction enzyme digestion M, DL5000 DNA marker; 1, Enzymatic cleavage of PrPP with SalⅠ and XmaⅠ; 2, rCMV; 3, pVAX1-1; 4, Enzymatic cleavage of prPgP with AflⅡ and NheⅠ; 5: GFP gene; 6: prPP.

Fig.5 Electrophoresis of PCR product of p△gErPgP-E2 plasmid target fragment M1, DL2000 DNA marker; 1, gE L; M2, DL1000 DNA marker; 2, gE R; 3, E2 gene.

Fig.6 Identification of p△gErPgP-E2 plasmid by double restriction enzyme digestion M, DL5000 DNA marker; 1, Enzymatic cleavage of p△gELrPgP with BamHⅠ and HindⅢ; 2, p△gELrPgP; 3, rCMV; 4, Enzymatic cleavage of p△gErPgP with PstⅠ and EcoRⅤ; 5, p△gELrPgP; 6, gE R; 7, Enzymatic cleavage of p△gErPgP-E2 with BglⅡ and PstⅠ; 8, p△gErPgP; 9, BVDV E2.

Fig.7 The expression of GFP in the process of recombinant virus screening (100×) A, C and E are the changes of green fluorescence of the 1st, 4th and 9th generation BHV-1 recombinant viruses in cell culture under fluorescent conditions; B, D and F are the cell morphology under bright field corresponding to A, C and E, respectively.

Fig.8 PCR identification of recombinant virus M1, DL500 DNA marker; 1, Identification of GFP in recombinant virus; 2, GFP positive control; 3, GFP negative control; M2, DL2000 DNA marker; 4, Deletion identification of gE fragment in recombinant virus; 5, gE positive control; 6, gE negative control; 7 and 8, Identification of E2 in recombinantvirus; 9 and 10, E2 positive control; 11, E2 negative control.

Table 4 Determination result of TCID50 of BHV-1

稀释倍数 Dilution multiple	病变数/总数 Lesion number/Total number	阳性占比 Positive ratio/%
10^-3	8/8	100
10^-4	8/8	100
10^-5	7/8	91.70
10^-6	4/8	50
10^-7	0/8	0
10^-8	0/8	0
阴性对照	0/8	0
Negative control

Table 5 Determination result of TCID50 of recombinant virus rBHV-1-△gE/E2 TCID50

稀释倍数 Dilution multiple	病变数/总数 Lesion number/Total number	阳性占比 Positive ratio/%
10^-3	8/8	100
10^-4	8/8	100
10^-5	6/8	75
10^-6	2/8	25
10^-7	0/8	0
10^-8	0/8	0
阴性对照	0/8	0
Negative control

Fig.9 Growth curve of recombinant virus and parent virus

Fig.10 IFA result of MDBK cell inoculated with recombinant virus (100×) A is the fluorescence of recombinant strain rBHV-1-ΔgE/E2 infected cells; C is the fluorescence of parental virus BHV-1 infected cells; B and D are the cell morphology under bright field corresponding to A and C, respectively.

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Construction and preliminary application of the universal transfer vector in recombination of Bovine herpes virus type 1

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