阅读说明：本技术 一种轮状病毒vp7重组蛋白及其应用 (Rotavirus VP7 recombinant protein and application thereof ) 是由 张莉莉 方荣祥 宋直钰 谢传淼 霍岩 张玉满 瞿维欢 刘超仁 于 2020-11-06 设计创作，主要内容包括：本发明公开了一种轮状病毒VP7重组蛋白及其应用,所述VP7重组蛋白为序列2所述的氨基酸序列的短截体,所述短截体不含序列2中第284-322位的序列。使用所述VP7重组蛋白(△VP7*)免疫兔获得的△VP7*抗体,检测其保护细胞抵抗轮状病毒RF毒株(G6P[1]型)侵染的中和抗体保护效价为1:21.97。本发明的VP7重组蛋白为通过该抗原设计重组亚单位疫苗,预防现阶段HRV流行株的感染提供了分子基础。(The invention discloses a rotavirus VP7 recombinant protein and application thereof, wherein the VP7 recombinant protein is a truncation of an amino acid sequence described in a sequence 2, and the truncation does not contain a sequence at the 284-322 th site in the sequence 2. The antibody Δ VP7 obtained by immunizing rabbits with the VP7 recombinant protein (Δ VP7) was tested for a protective titer of neutralizing antibody against infection of rotavirus RF strain (G6P [1 ]) of 1: 21.97. The VP7 recombinant protein provides a molecular basis for designing a recombinant subunit vaccine through the antigen and preventing the infection of HRV epidemic strains at the present stage.)

1. The application of the protein in any one of the following P1-P3:

the application of the protein in preparing anti-rotavirus serum;

p2. the use of said protein in the preparation of rotavirus antibodies;

the application of the protein in preparing anti-rotavirus vaccine;

the protein is A1) or A2):

A1) VP7 recombinant protein, wherein the VP7 recombinant protein is a short truncation of the amino acid sequence described in sequence 2, and the short truncation does not contain the sequence at position 284-322 in sequence 2;

A2) a1) at the N-terminus or/and the C-terminus.

2. The use of claim 1, wherein the amino acid sequence of the VP7 recombinant protein is the sequence shown at positions 51-283 in SEQ ID NO 2.

3. Use of a biomaterial related to a protein according to claim 1 or 2 in any one of the following Q1-Q3:

q1. application of the biological material in preparing anti-rotavirus serum;

the application of the biological material in preparing rotavirus antibodies;

q3. the use of the biomaterial in the manufacture of a vaccine against rotavirus;

the biological material is shown in any one of the following B1) -B5):

B1) a nucleic acid molecule encoding the protein of claim 1 or 2;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic animal cell line containing the nucleic acid molecule according to B1) or a transgenic animal cell line containing the expression cassette according to B2).

4. The use as claimed in claim 3, wherein B1) is a DNA molecule represented by position 151-849 in sequence 1 of the sequence Listing.

5. A protein, wherein said protein is a1) or a 2):

A1) VP7 recombinant protein, wherein the VP7 recombinant protein is a short truncation of the amino acid sequence described in sequence 2, and the short truncation does not contain the sequence at position 284-322 in sequence 2;

A2) a1) at the N-terminus or/and the C-terminus.

6. The protein of claim 5, wherein the amino acid sequence of the VP7 recombinant protein is the sequence shown in positions 51-283 of SEQ ID NO 2.

7. The biological material related to the protein of claim 5 or 6 is any one of the following B1) -B5):

B1) a nucleic acid molecule encoding the protein of claim 5 or 6;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic animal cell line containing the nucleic acid molecule according to B1) or a transgenic animal cell line containing the expression cassette according to B2).

8. The biomaterial as claimed in claim 7, wherein B1) is a DNA molecule represented by position 151-849 in sequence 1 of the sequence Listing.

9. A method for preparing anti-rotavirus serum, comprising the step of immunizing an animal with the protein of claim 5 or 6 to obtain anti-rotavirus serum.

10. The use of the preparation method of claim 9 in any one of the following R1-R3:

use of the preparation method of claim 9 in the preparation of anti-rotavirus serum;

use of the preparation method of claim 9 for the preparation of rotavirus antibodies;

use of the preparation method of claim 9 in the preparation of vaccines against rotavirus.

Technical Field

The invention belongs to the technical field of biology, and relates to a rotavirus delta VP7 non-cytotoxic recombinant protein and application thereof.

Background

Rotavirus (RV) is a main cause of severe diarrhea of infants, about hundreds of thousands of cases of infant death are caused each year, and vaccination is the only effective prevention and control means. RV vaccines currently on the market are all oral live attenuated vaccines, which are listed by the WHO as "vaccines with process needs improvement" due to their potential risk of intussusception. The subunit vaccine is prepared by taking protective antigen of virus as a core and adjuvant, has good safety and can stimulate the organism to generate enough immunity. Meanwhile, the subunit vaccine can realize accurate immunity to the circulating strains and deal with the rapid variation of RV.

The major neutralizing antigens of RV are VP7(G protein) and VP4(P protein), and at least 27G genotypes have been identified. The molecular flow of human rotavirus RV (HRV) in China is staged, G1 is the main epidemic G type (74.3%) before 2000, G3 (45.2%) is the main epidemic G type, and G1 (21.3%) is the main epidemic G type after 2000 and 2012. From 2012 onwards, RV type G9 was identified as the currently predominant strain prevalent in China. It is worth noting that according to the clade characteristics of the VP7 gene (G9-I-G9-VI), the current epidemic strain is G9-VI (accounting for 96.5%), which is different from the G9-I in the early nineties in China and the widely epidemic G9-III in foreign countries. Whether these circulating strains alter the pathogenicity of humans and whether existing vaccines produce good protection needs further investigation. Designing subunit vaccines according to the VP7 sequence of an epidemic strain is one of the strategies for realizing accurate immunity, but the development process is limited by cytotoxicity caused by full-length expression of the VP7 gene.

Disclosure of Invention

The invention aims to obtain a nontoxic delta VP7 sequence from escherichia coli by taking an HRV epidemic strain BJ-Q794 of Beijing at the present stage as a target strain, thereby providing a potential candidate vaccine for HRV prevention and control.

The invention provides a protein, which is A1) or A2):

A1) VP7 recombinant protein, wherein the VP7 recombinant protein is a short truncation of the amino acid sequence described in sequence 2, and the short truncation does not contain the sequence at position 284-322 in sequence 2;

A2) a1) at the N-terminus or/and the C-terminus.

The amino acid sequence of the VP7 recombinant protein is shown as 51-283 in the sequence 2.

The invention also provides a biomaterial related to the VP7 recombinant protein, which is shown in any one of the following B1) -B5):

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic animal cell line containing the nucleic acid molecule according to B1) or a transgenic animal cell line containing the expression cassette according to B2).

B1) is a DNA molecule shown in the 151 th-849 th site of the sequence 1 in the sequence table.

The application of the protein disclosed by the invention in any one of the following P1-P3 also belongs to the protection scope of the invention:

the application of the protein in preparing anti-rotavirus serum;

p2. the use of said protein in the preparation of rotavirus antibodies;

and P3, the protein is applied to the preparation of vaccines against rotavirus.

The application of the biological material related to the protein in the invention in any one of the following Q1-Q3 also falls within the protection scope of the invention:

q1. application of the biological material in preparing anti-rotavirus serum;

the application of the biological material in preparing rotavirus antibodies;

q3. application of the biological material in preparing vaccines against rotavirus.

The invention provides a preparation method of anti-rotavirus serum, which comprises the step of immunizing animals by using the protein to obtain the anti-rotavirus serum.

The application of the preparation method of the anti-rotavirus serum in any one of the following R1-R3 also belongs to the protection scope of the invention:

r1. the application of the preparation method in preparing anti-rotavirus serum;

r2. the preparation method is applied to the preparation of rotavirus antibodies;

and R3, the preparation method is applied to preparation of rotavirus resisting vaccines.

The invention has the beneficial effects that: a recombinant protein VP7, a truncation of a main antigen of an HRV epidemic strain, is provided, the recombinant protein VP7 has no cytotoxicity, and the protective titer of a neutralizing antibody for protecting cells from infection of rotavirus RF strain (G6P [1 ]) is detected to be 1:21.97 by using an antibody delta VP7 obtained by immunizing rabbits with the recombinant protein VP7 (delta VP 7). The VP7 recombinant protein provides a molecular basis for designing a recombinant subunit vaccine through the antigen and preventing the infection of HRV epidemic strains at the present stage.

Drawings

FIG. 1 Western results of GST (glutathione mercaptotransferase) induced expression.

FIG. 2 Western results of expression induction by Transetta (DE3) -. DELTA.VP 7-1.

FIG. 3 Western results of expression induction by Transetta (DE3) -. DELTA.VP 7-9.

FIG. 4 Western results of expression induction by Transetta (DE3) -. DELTA.VP 7-10, and by Transetta (DE3) -. DELTA.VP 7-11.

FIG. 5 Western results of inducible expression of Transetta (DE3) -. DELTA.VP 7-12.

FIG. 6 Western results of inducible expression by Transetta (DE3) -. DELTA.VP 7-15 and Transetta (DE3) -. DELTA.VP 7-16.

FIG. 7 is a schematic diagram of Δ VP7, gray for non-toxic recombinant protein and black for toxic recombinant protein, wherein the toxic region is 284-322 amino acid fragment.

FIG. 8 Western results of the induction of expression of recombinant protein Δ VP 7.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction of VP7 and Δ VP7

The complete gene sequence of VP7 of BJ-Q794 is synthesized (the sequence synthesis is entrusted to Shanghai Jie biological engineering Co., Ltd.), the amino acid sequence of VP7 is shown as the sequence 2 in the sequence table, and the complete gene (nucleotide) sequence of VP7 is shown as the sequence 1 in the sequence table. The entire gene sequence of VP7 was inserted between SmaI and SalI sites of pGH vector (Shanghai Czejust bioengineering, Ltd.) while keeping the other sequences of pGH vector unchanged to obtain recombinant plasmid pGH-J1-VP 7. HRV epidemic strain BJ-Q794 (genotype G9P [8], GenBank sequence number: KF673438) is the target strain (reference: royal jelly et al, G9-VI rotavirus reappeared and predominance in children with Beijing diarrhea, Virol, 2016,32 (4): DOI:10.13242/j.cnki. bindduxuebao.002983).

According to the structural analysis of VP7 protein, a series of truncations are designed, which are respectively delta VP7-1, delta VP7-2, delta VP7-3, delta VP7-4, delta VP7-5, delta VP7-6, delta VP7-7, delta VP7-8, delta VP7-9, delta VP7-10, delta VP7-11, delta VP7-12, delta VP7-13, delta VP7-14, delta VP7-15, delta VP7-16, delta VP7-17 and delta VP7, wherein the amino acid sequence of delta VP7-1 is the 55-326 th position of sequence 1, the amino acid sequence of VP7-2 is the 51-326 th position of sequence 1, the amino acid sequence of delta VP7-3 is the 58-326 th position of sequence 1, the amino acid sequence of delta 7-4 is the 51-326 th position of sequence 865-327 of sequence 861, the amino acid sequence of the delta VP7-6 is the 109-326 th position of the sequence 1, the amino acid sequence of the delta VP7-7 is the 51-322 th position of the sequence 1, the amino acid sequence of the delta VP7-8 is the 109-215 th position of the sequence 1, the amino acid sequence of the delta VP7-9 is the 216-322 th position of the sequence 1, the amino acid sequence of the delta VP7-10 is the 216-265 th position of the sequence 1, the amino acid sequence of the delta VP7-11 is the 266-322 th position of the sequence 1, the amino acid sequence of the delta VP7-12 is the 266-322 th position of the sequence 1, the amino acid sequence of the delta VP7-13 is the 293-322 th position of the sequence 1, the amino acid sequence of the delta VP7-14 is the 284-322 th position of the sequence 1, the amino acid sequence of the delta VP7-15 is the 284-304 th position of the sequence 1, and the amino acid sequence of the delta VP7-16 is the 284-312 st position of the 284-312, the amino acid sequence of delta VP7-17 is 51-265 th position of sequence 1, the amino acid sequence of delta VP7 is 51-283 th position of sequence 1, and the designed primers corresponding to the above-mentioned short truncations are shown in Table 1. Recombinant plasmid pGH-J1-VP7 is used as a template, and primer pairs in Table 1 are respectively used as primers for amplification to obtain amplification products VP7, delta VP7-1, delta VP7-2, delta VP7-3, delta VP7-4, delta VP7-5, delta VP7-6, delta VP7-7, delta VP7-8, delta VP7-9, delta VP7-10, delta VP7-11, delta VP7-12, delta VP7-13, delta VP7-14, delta VP7-15, delta VP7-16, delta VP7-17 and delta VP 7. The resulting amplified products were used to replace the sequence between the XmaI and EcoRI recognition sites in pGEX-3X vector (GE Co.) and the other sequences of pGEX-3X vector were kept unchanged, resulting in recombinant vectors pGEX-3X-VP7, pGEX-3X- Δ VP7-1, pGEX-3X- Δ VP7-2, pGEX-3X- Δ VP7-3, pGEX-3X- Δ VP7-4, pGEX-3X- Δ VP7-5, pGEX-3X- Δ VP7-6, pGEX-3X- Δ VP7-7, pGEX-3X- Δ VP7-8, pGEX-3X-VP 7-9, pGEX-3X- Δ VP7-10, pGEX-3X-VP 7-11, pGEX-3X-7-12, pGEX-3X-delta VP7-13, pGEX-3X-delta VP7-14, pGEX-3X-delta VP7-15, pGEX-3X-delta VP7-16, pGEX-3X-delta VP7-17 and pGEX-3X-delta VP7, each recombinant vector can express a fusion protein formed by fusing a VP7 protein or a corresponding VP7 protein truncation at the N end with a GST tag; the recombinant vectors pGEX-3X-VP7, pGEX-3X-delta VP7-1, pGEX-3X-delta VP7-2, pGEX-3X-delta VP7-3, pGEX-3X-delta VP7-4, pGEX-3X-delta VP7-5, pGEX-3X-delta VP7-6, pGEX-3X-delta VP7-7, pGEX-3X-delta VP7-8, pGEX-3X-delta VP7-9, pGEX-3X-delta VP7-10, pGEX-3X-delta VP7-11, pGEX-3X-VP 7-12, pGEX-3X-delta VP7-13, pGEX-3X-delta VP7-14, pGEX-3X-delta VP7-15, pGEX-3X- Δ VP7-16, pGEX-3X- Δ VP7-17 and pGEX-3X- Δ VP7 Escherichia coli Trans10 (Beijing Quanjin Biotechnology Co., Ltd.) was transformed to obtain a series of recombinant plasmids carrying the correct Δ VP7 sequence, each of which was transformed into Escherichia coli Transetta (DE3) (Beijing Quanjin Biotechnology Co., Ltd.) and was Escherichia coli Transetta (DE3) -VP7, Transetta (DE3) -. DELTA VP7-1, Transetta (DE3) -. DELTA VP 42-2, Transetta (DE3) -. DELTA VP7-3, Transetta (DE3) -. VP7-4, Transetta (DE3) -. DELTA. 7-5, Transetta (DE 3. DELTA.). VP7-6, Transetta (DE-3) -. DELTA.) -7, Transetta (VP 363672) -. DELTA.) -363672, and a sequence of Escherichia coli Transetta, Transetta (DE3) -. DELTA.VP 7-10, Transetta (DE3) -. DELTA.VP 7-11, Transetta (DE3) -. DELTA.VP 7-12, Transetta (DE3) -. DELTA.VP 7-13, Transetta (DE3) -. DELTA.VP 7-14, Transetta (DE3) -. DELTA.VP 7-15, Transetta (DE3) -. DELTA.VP 7-16, Transetta (DE3) -. DELTA.VP 7-17 and Transetta (DE3) -. DELTA.VP 7, which are used as host bacteria for inducible expression of fusion proteins.

TABLE 1 PCR primers expressing VP7 and Δ VP7

Example 2 inducible expression of the VP7 and Δ VP7 fusion proteins

1. Escherichia coli Transetta (DE3) -VP7 prepared in example 1 was cultured in LB liquid medium at 220rpm at 37 ℃ overnight (16 hours). Transferred into 500ml of fresh medium according to the ratio of 1:1000Continuing to culture to OD₆₀₀Between about 0.35 and 0.5, isopropyl-. beta. -D-thiogalactoside (IPTG) was added to induce at a final concentration of 0.1 mM. The method for inducing expression of the fusion protein comprises the following steps: the cells were cultured at 37 ℃ and 220rpm for 4 hours under induction, and the precipitated cells were collected by centrifugation at 4 ℃ and prepared for extraction of the fusion protein.

2. The method for inducing expression of the fusion protein in the step 1 is replaced by the following steps: the cells were cultured overnight (16 hours) at 16 ℃ and 180rpm, and the precipitated cells were collected by centrifugation at 4 ℃ and prepared for extraction of the fusion protein. The other steps are unchanged.

3. Adding PBS buffer solution into the thallus obtained in the step 1 or 2 for fully suspending, adding a loading buffer solution (Bio-rad, cat number: 1610747) of 4 xSDS-PAGE, boiling in water bath for 5min for lysing the thallus, centrifugally collecting supernatant containing the fusion protein, performing gel separation on the protein through SDS-PAGE, and detecting the protein expression condition through Western blots. The primary antibody of Western blots was Rabbit polyclonal to GST (Abcam, cat # ab19256) at 1:1000 dilution. The secondary antibody was Anti-Rabbit IgG (H + L), HRP Conjugate (Promega, cat # W4011), 1:1000 dilution. The development was carried out using a chemiluminescent substrate (Thermo Fisher, SuperSignal West Dura, cat. No.: A38555).

The Escherichia coli Transetta (DE3) -VP7 in the steps 1-3 is sequentially replaced by Escherichia coli Transetta (DE3) -. DELTA.VP 7-1, Transetta (DE3) -. DELTA.VP 7-2, Transetta (DE3) -. DELTA.VP 7-3, Transetta (DE3) -. DELTA.VP 7-4, Transetta (DE3) -. DELTA.VP 7-5, Transetta (DE3) -. DELTA.VP 7-6, Transetta (DE3) -. DELTA.VP 7-7, Transetta (DE3) -. DELTA.VP 7-8, Transetta (DE3) -. DELTA.VP 7-9, transetta (DE3) -. DELTA.VP 7-10, Transetta (DE3) -. DELTA.VP 7-11, Transetta (DE3) -. DELTA.VP 7-12, Transetta (DE3) -. DELTA.VP 7-13, Transetta (DE3) -. DELTA.VP 7-14, Transetta (DE3) -. DELTA.VP 7-15, Transetta (DE3) -. DELTA.VP 7-16, Transetta (DE3) -. DELTA.VP 7-17 or Transetta (DE3) -. DELTA.VP 7). The other steps are unchanged.

The results are shown in FIGS. 1 to 6, and FIG. 1 shows the Western blot results of GST (glutathione mercaptotransferase) -induced expression. Wherein the bacterial cells (Transetta (DE3) -VP7) are induced at 37 ℃ and the primary antibody is GST antibody. The arrow indicates the GST protein, 26.6kDa in size. Lanes 1, 2 are: not inducing thallus holoprotein, inducing thallus holoprotein. FIG. 2 shows the Western blot results of inducible expression of Transetta (DE3) -. DELTA.VP 7-1. In the A and B panels, the cells were induced at 37 deg.C (A) and 16 deg.C (B), respectively, and the primary antibody was GST antibody. The arrow indicates the fusion protein Δ VP7-1 fused to the GST tag, with a size of 57.5 kDa. Lanes 1-6 are: non-induced thallus soluble protein, non-induced thallus insoluble protein, non-induced thallus total protein, induced thallus soluble protein, induced thallus insoluble protein and induced thallus total protein. FIG. 3 shows the Western blot results of Transetta (DE3) -. DELTA.VP 7-9 induced expression. Wherein the bacteria are induced at 37 deg.C and 16 deg.C respectively, and the primary antibody is GST antibody. The arrow indicates a 38.8kDa Δ VP7-9 fusion protein fused to a GST tag. Lanes 1-4 are: the whole protein of the thallus is not induced at 37 ℃, the whole protein of the thallus is not induced at 16 ℃ and the whole protein of the thallus is induced at 16 ℃. FIG. 4 shows Western blot results of inducible expression by Transetta (DE3) -. DELTA.VP 7-10 and Transetta (DE3) -. DELTA.VP 7-11. Wherein the bacteria are induced at 37 deg.C and 16 deg.C respectively, and the primary antibody is GST antibody. Arrows in lanes 1-4 show the Δ VP7-10 fusion protein fused to the GST tag, with a size of 32.0 kDa. Arrows in lanes 5-8 show the Δ VP7-11 fusion protein fused to the GST tag, with a size of 32.0 kDa. Lanes 1-4, 5-8 are: the whole protein of the thallus is not induced at 37 ℃, the whole protein of the thallus is not induced at 16 ℃ and the whole protein of the thallus is induced at 16 ℃. FIG. 5 shows the Western blot results of inducible expression of Transetta (DE3) -. DELTA.VP 7-12. Wherein the bacteria are induced at 37 deg.C and 16 deg.C respectively, and the primary antibody is GST antibody. The arrow indicates the fusion protein Δ VP7-12 fused to the GST tag, which is 29.8kDa in size. Lanes 1-3 are: not inducing thallus holoprotein at 37 ℃, inducing thallus holoprotein at 37 ℃ and inducing thallus holoprotein at 16 ℃. FIG. 6 shows Western blot results of inducible expression by Transetta (DE3) -. DELTA.VP 7-15 and Transetta (DE3) -. DELTA.VP 7-16. Wherein the bacteria are induced at 37 deg.C and 16 deg.C respectively, and the primary antibody is GST antibody. Arrows in lanes 1-3 show the Δ VP7-15 fusion protein fused to the GST tag, and has a size of 29.4 kDa. Arrows in lanes 4-6 show the Δ VP7-16 fusion protein fused to the GST tag, with a size of 30.3 kDa. Lanes 1-3, 4-6 are: not inducing thallus holoprotein at 37 ℃, inducing thallus holoprotein at 37 ℃ and inducing thallus holoprotein at 16 ℃. Lane 7 is the GST-induced whole cell protein at 37 ℃

After the VP7 and each delta VP7 fusion protein are induced to express, the growth of the host bacteria is judged for cytotoxicity. OD of host bacteria before and after induction expression of VP7 and each delta VP7 fusion protein₆₀₀The measurements are shown in Table 2:

TABLE 2 various Δ G9P [8]]Post-induction OD of VP7 protein₆₀₀

As can be seen from the results in Table 2, only the Δ VP7-8(109-₆₀₀Significantly increased) indicating that the short truncates Δ VP7-8(109-215), Δ VP7-10(216-265), Δ VP7-12(266-292) and Δ VP7 (51-283) are not cytotoxic, and that the other short truncates are cytotoxic, as shown in FIG. 7. FIG. 7 shows the respective Δ VP7 truncated sequences, wherein grey indicates the non-toxic recombinant protein and black indicates the toxic recombinant protein, wherein the toxic region is the 284-322 amino acid fragment.

Example 3 purification of Δ VP7 fusion protein, antibody preparation and antibody titer detection

1. Escherichia coli prepared in example 1 was cultured in Transetta (DE3) -. DELTA.VP 7 in LB liquid medium at 220rpm at 37 ℃ overnight (16 hours). Transferring the culture medium into a fresh culture medium according to the ratio of 1:1000, and continuing culturing until OD is reached₆₀₀Between about 0.35 and 0.5, isopropyl-. beta. -D-thiogalactoside (IPTG) was added to induce at a final concentration of 0.1 mM. The method for inducing expression comprises the following steps: the cells were cultured overnight (16 hours) at 16 ℃ and 180rpm, and the precipitated cells were collected by centrifugation at 4 ℃ and prepared for extraction of the fusion protein Δ VP 7.

2. The cells (500ml) were collected by centrifugation, resuspended in PBS containing protease inhibitors, the supernatant removed by centrifugation, resuspended again in 20ml PBS containing protease inhibitors and placed on ice.

3. The thalli is crushed by a low-temperature ultrahigh-pressure continuous flow cell crusher.

4. The soluble protein was collected by centrifugation.

5. Protein purification was performed by GST tag, protein was purified by a Glutathione Sepharose 4 Fast Flow (GE Healthcare, 17-5132-01) gel column, target protein was eluted with 50mM Tris-HCl pH 8.0 containing 20mM reduced Glutathione, target protein was concentrated by ultrafiltration, and the protein storage solution was changed to PBS solution, thereby obtaining GST-. DELTA.VP 7 solution. The protein purity was 85% and was quantified by bradford assay.

The fusion protein (GST-delta VP7) expressed in E.coli was fused with the N-terminal GST tag by the delta VP7 (26 kDa) shown in positions 51-283 of SEQ ID NO. 2, and was 26.88kDa in size and 52.58kDa in size of GST-delta VP 7. GST- Δ VP7 was 85% pure, as shown in FIG. 8. 500ml of E.coli suspension was purified to obtain 0.75mg of GST- Δ VP 7-fusion protein.

6. Purified GST-delta VP7 immune rabbit (animal technology limited, Kyowa laboratory, Beijing) was used to prepare antibody (immune experiment was performed in the center of the institute of genetics and developmental biology, China academy of sciences), immunization was performed in four times, the first immunization was performed with a mixed solution obtained by mixing 1:1 volume of GST-delta VP7 obtained in step 5 with complete Freund's adjuvant (Sigma Co.), the amount of GST-delta VP7 was 0.5mg, the second immunization was performed with a mixed solution obtained by mixing 1:1 volume of incomplete Freund's adjuvant (Sigma Co.) with the GST-delta VP7 obtained in step 5, the amount of GST-delta VP7 was 0.25mg, the third immunization was performed with 1:1 volume of incomplete Freund's adjuvant (Sigma Co.), the mixed solution obtained by mixing 1:1 volume of GST-delta VP7 obtained in step 5 with GST-delta VP7 mg, the amount of GST-delta VP7 mg, the third immunization was performed with 1:1 volume of incomplete GST-delta VP7 mg Delta VP7 solution was mixed to obtain a mixture, GST-delta VP7 was used in an amount of 0.5mg, the time interval between immunizations was 14 days, and blood was taken from carotid artery by intubation 3 weeks after the last immunization to obtain serum.

7. The resulting serum was tested for neutralizing antibody titer:

1) MA104 cells (monkey fetal kidney cells) were passaged into 96-well plates and cultured for 16-20 hours.

2) Test serum was taken, subjected to water bath at 60 ℃ for 30min to inactivate complement, and then diluted 4-fold in a gradient in MEM medium containing 2. mu.g/ml of pancreatin to give each serum dilution.

3) Taking rotavirus RF strain (G6P [1]]Type) (described in James E Richards, Ulrich Desselberger, Andrew M Lever. Experimental products of surgery reduction a rotaviruses genes system: synthetic full length peptides of surgery were each isolated in vitro cells plos one.8(9): e74328, 2013.) EDTA-free pancreatic enzyme was added to a concentration of 10. mu.g/ml, followed by incubation at 37 ℃ for 1 hour and then diluted to 200TCID with MEM medium containing 2. mu.g/ml pancreatic enzyme₅₀。

4) Mixing the serum diluent obtained in the step 2) and the virus solution obtained in the step 3) in equal volume, and incubating for 1 hour at 37 ℃.

5) After completion of step 1), the cells were washed 3 times with MEM medium, and then 20. mu.l of the mixture obtained in step 4) was added to each well and incubated at 37 ℃ for 1 hour.

6) After completion of step 5), the supernatant was aspirated, and MEM medium containing 2. mu.g/ml of trypsin was added and cultured at 37 ℃ for 3 days.

7) After completion of step 6), the cytopathic condition was observed and the serum neutralizing antibody titer was calculated.

lg(TCID₅₀) Logarithm-distance ratio of greater than 50% serum dilution x logarithm of dilution factor ═ n

TCID₅₀＝10ⁿ,20μl

10ⁿA serum that is 1: m, i.e. 1: m, protects the cells from disease, i.e. the neutralizing antibody titer of that serum.

The delta VP7 antibody obtained from immunized rabbit, the protective titer of neutralizing antibody for protecting cell from infection of rotavirus RF strain (G6P [1 ]) is 1: 21.97.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> plant chain (Shanghai) Biotechnology Ltd

<120> rotavirus VP7 recombinant protein and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 981

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtatggta ttgaatatac cacagttcta acctttctga tatcaatagt tttattgaac 60

tacatattaa aatcactaac tagtgcgatg gactttatac tttatagatt tcttttactt 120

attgttattt tgtcgccatt tgtcaaaaca caaaattatg ggataaattt accaattact 180

ggctccatgg acacagtata tgcaaattcg tcacagcaag aaacattttt aacttcaacg 240

ctatgtttat attatcctac tgaagcatca actcaaattg gagatactga atggaagaat 300

actctatctc aattattctt gactaagggg tggccaactg gatcagtcta ttttaaagaa 360

tatacagata tcgcttcatt ctcaattgat ccacaacttt attgtgatta taatgttgtg 420

ctaatgaagc atgattcaac gttagagcta gatatgtcag aattggctga tttgattcta 480

aatgaatggt tatgcaatcc aatggatata acattatatt attatcagca aacagatgaa 540

tcgaataaat ggatatcgat gggacaatct tgtaccataa aagtgtgccc attaaataca 600

caaactttag gaataggttg tactactaca aatacagcga catttgaaga agtagctact 660

agtgagaaat tagtgataac tgatgttgtt gatggcgtga atcataagct tgatgtaact 720

acaaatacct gtacaattag aaattgtaag aagttaggac cgagagaaaa tgtagcaatt 780

atacaagtcg gtggctcaga agtgttagat attacagcgg atccaactac cacaccacaa 840

actgagcgta tgatgcgagt aaattggaag aaatggtggc aagttttcta tacagtagta 900

gattacatta atcagattgt gcaatttatg tccaaaagat cacggtcatt aaattcagca 960

gctttttatt atagagtctg a 981

<210> 2

<211> 326

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Tyr Gly Ile Glu Tyr Thr Thr Val Leu Thr Phe Leu Ile Ser Ile

1 5 10 15

Val Leu Leu Asn Tyr Ile Leu Lys Ser Leu Thr Ser Ala Met Asp Phe

20 25 30

Ile Leu Tyr Arg Phe Leu Leu Leu Ile Val Ile Leu Ser Pro Phe Val

35 40 45

Lys Thr Gln Asn Tyr Gly Ile Asn Leu Pro Ile Thr Gly Ser Met Asp

50 55 60

Thr Val Tyr Ala Asn Ser Ser Gln Gln Glu Thr Phe Leu Thr Ser Thr

65 70 75 80

Leu Cys Leu Tyr Tyr Pro Thr Glu Ala Ser Thr Gln Ile Gly Asp Thr

85 90 95

Glu Trp Lys Asn Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro

100 105 110

Thr Gly Ser Val Tyr Phe Lys Glu Tyr Thr Asp Ile Ala Ser Phe Ser

115 120 125

Ile Asp Pro Gln Leu Tyr Cys Asp Tyr Asn Val Val Leu Met Lys His

130 135 140

Asp Ser Thr Leu Glu Leu Asp Met Ser Glu Leu Ala Asp Leu Ile Leu

145 150 155 160

Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Thr Leu Tyr Tyr Tyr Gln

165 170 175

Gln Thr Asp Glu Ser Asn Lys Trp Ile Ser Met Gly Gln Ser Cys Thr

180 185 190

Ile Lys Val Cys Pro Leu Asn Thr Gln Thr Leu Gly Ile Gly Cys Thr

195 200 205

Thr Thr Asn Thr Ala Thr Phe Glu Glu Val Ala Thr Ser Glu Lys Leu

210 215 220

Val Ile Thr Asp Val Val Asp Gly Val Asn His Lys Leu Asp Val Thr

225 230 235 240

Thr Asn Thr Cys Thr Ile Arg Asn Cys Lys Lys Leu Gly Pro Arg Glu

245 250 255

Asn Val Ala Ile Ile Gln Val Gly Gly Ser Glu Val Leu Asp Ile Thr

260 265 270

Ala Asp Pro Thr Thr Thr Pro Gln Thr Glu Arg Met Met Arg Val Asn

275 280 285

Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr Val Val Asp Tyr Ile Asn

290 295 300

Gln Ile Val Gln Phe Met Ser Lys Arg Ser Arg Ser Leu Asn Ser Ala

305 310 315 320

Ala Phe Tyr Tyr Arg Val

325